Nucleic acid fragments encoding isoflavone synthase

ABSTRACT

This invention relates to an isolated nucleic acid sequence encoding isoflavone synthase. The invention also relates to the construction of chimeric sequences encoding all or a substantial portion of the enzymes, in sense or antisense orientation, wherein expression of the chimeric sequence results in production of altered levels of the enzyme in a transformed host cell.

This application claims the benefit of U.S. Provisional Application No.60/117,769, filed Jan. 27, 1999, U.S. Provisional Application No.60/144,783, filed Jul. 20, 1999, and U.S. Provisional Application No.60/156,094, filed Sep. 24, 1999.

FIELD OF THE INVENTION

This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid sequences encodingisoflavone synthase and their use in producing isoflavones.

BACKGROUND OF THE INVENTION

Isoflavonoids represent a class of secondary metabolites produced inlegumes by a branch of the phenylpropanoid pathway and include suchcompounds as isoflavones, isoflavanones, rotenoids, pterocarpans,isoflavans, quinone derivatives, 3-aryl-4-hydroxy-coumarins,3-arylcoumarins, isoflav-3-enes, coumestans, alpha-methyldeoxybenzoins,2-arylbenzofurans, isoflavanol, coumaronochromone and the like. Inplants, these compounds are known to be involved in interactions withother organisms and to participate in the defense responses of legumesagainst phytopathogenic microorganisms (Dewick, P. M. (1993) in TheFlavonoids, Advances in Research Since 1986, Harborne, J. B. Ed., pp.117-238, Chapman and Hall, London). Isoflavonoid-derived compounds alsoare involved in symbiotic relationships between roots and rhizobialbacteria which eventually result in nodulation and nitrogen-fixation(Phillips, D. A. (1992) in Recent Advances in Phytochemistry. Vol. 26,pp 201-231, Stafford, H. A. and Ibrahim, R. K., Eds, Pleneum Press, NewYork), and overall they have been shown to act as antibiotics,repellents, attractants, and signal compounds (Barz, W. and Welle, R.(1992) Phenolic Metabolism in Plants, pg 139-164, Ed by H. A. Staffordand R. K. Ibrahim, Plenum Press, New York).

Isoflavonoids have also been reported to have physiological activity inanimal and human studies. For example, it has been reported that theisoflavones found in soybean seeds possess antihemolytic (Naim, M., etal. (1976) J. Agric. Food Chem. 24:1174-1177), antifungal (Naim, M., etal. (1974) J. Agr. Food Chem. 22:806-810), estrogenic (Price, K. R. andFenwick, G. R. (1985) Food Addit. Contam. 2:73-106), tumor-suppressing(Messina, M. and Barnes, S. (1991) J. Natl. Cancer Inst. 83:541-546;Peterson, G., et al. (1991) Biochem. Biophys. Res. Commun. 179:661-667),hypolipidemic (Mathur, K., et al. (1964) J. Nutr. 84:201-204), and serumcholesterol-lowering (Sharma, R. D. (1979) Lipids 14:535-540) effects.These epidemiological studies indicate that isoflavones in soybeanprotein products, when taken as a dietary supplement, may produce manysignificant health benefits.

Free isoflavones rarely accumulate to high levels in soybeans. Insteadthey are usually conjugated to carbohydrates or organic acids. Soybeanseeds contain three types of isoflavones in four different forms: theaglycones, daidzein, genistein and glycitein; the glucosides, daidzin,genistin and glycitin; the acetylgucosides, 6″-O-acetyldaidzin,6″-O-acetylgenistin and 6″-O-acetylglycitin; and the malonylglucosides,6″-O-malonyldaidzin, 6″-O-malonylgenistin and 6″-O-malonylglycitin. Inaccordance with the present invention, all of these compounds areincluded in the term isoflavonoids. The content of isoflavonoids insoybean seeds is quite variable and is affected by both genetics andenvironmental conditions such as growing location and temperature duringseed fill (Tsukamoto, C., et al. (1995) J. Agric. Food Chem.43:1184-1192; Wang, H. and Murphy, P. A. (1994) J. Agric. Food Chem.42:1674-1677). In addition, isoflavonoid content in legumes can bestress-induced by pathogenic attack, wounding, high UV light exposureand pollution (Dixon, R. A. and Paiva, N. L. (1995) Plant Cell7:1085-1097).

The biosynthetic pathway for isoflavonoids in soybean and theirrelationship with several other classes of phenylpropanoids is presentedin FIG. 1. Many of the enzymes involved in the synthesis ofisoflavonoids in legumes have been identified and many of the genes inthe pathway have been cloned. These include three P450-dependentmonooxygenases, cinnamate 4-hydoxylase (Potts, J. R. M., et al. (1974)J. Biol. Chem. 249:5019-5026), isoflavone 2′-hydroxylase (Akashi, T. etal. (1998) Biochem. Biophys. Res. Commun. 251:67-70), anddihydroxypterocarpan 6a-hydroxylase (Schopfer, C. R., et. al. (1998)FEBS Lett. 432:182-186). However, to date the gene encoding isoflavonesynthase, the first step in the phenylpropanoid branch that commitsmetabolic intermediates to the synthesis of isoflavonoids, has beenneither identified nor cloned from any species. In this centralreaction, 2S-flavanone is converted into an isoflavonoid such asgenistein and daidzein. The enzymatic reaction for this oxidative arylmigration step was first reported by Hagmann, M. L. and Grisebach, H.((1984) FEBS Lett. 175:199-202). The reaction involves a P450monoxygenase-mediated conversion of the 2S-flavanone to a2-hydroxyisoflavanone, followed by conversion to the isoflavonoid. Thislast step is possibly mediated by a soluble dehydratase (Kochs, G. andGrisenbach, H. (1985) Eur. J. Biochem. 155:311-318). However, the2-hydroxyisoflavanone intermediate was described as unstable and couldconvert directly to genistein.

Cytochrome P450-dependant monooxygenases comprise a large group ofheme-containing enzymes, most of which catalyze NADPH- and O₂-dependanthydroxylation reactions. Most of these enzymes do not use NADPHdirectly, but rely upon an interaction with a flavoprotein known as aP450 reductase that transfers electrons from the cofactor to the P450.Cloning of plant P450s by traditional protein purification strategieshas been difficult, as these membrane-bound proteins are often veryunstable and are typically present in low abundance. PCR-based cloningstrategies using sequence homologies between P450s has increaseddramatically the number of P450 genes cloned. However, the in vivoactivity of many of these cloned genes remains unknown and they areclassified simply as P450s, and are grouped into families based solelyon sequence homology (Chapple, C. (1998) Annu. Rev. Plant Physiol. PlantMol. Bio. 49:311-343). Proteins that are greater than 55% identical aredesignated as members of the same subfamily, while P450s that are 97%identical, or greater, are assumed to be allelic variants of the samegene (Chapple, C. (1998) Annu. Rev. Plant Physiol. Plant Mol. Bio.49:311-343).

Efforts to determine in vivo activities of existing P450 clones areincreasing. Most efforts involve expressing genes or cDNAs for P450s inyeast or insect cell systems, and then screening for a particularactivity. For example, isoflavone 2′-hydroxylase (Akashi, T., et al.(1998) Biochem. Biophys. Res. Commun. 251:67-70) anddihydroxypterocarpan 6a-hydroxylase (Schopfer, C. R., et al. (1998) FEBSLetters 432:182-186) were identified in this manner.

The physiological activities associated with isoflavonoids in bothplants and humans makes the manipulation of their contents in cropplants highly desirable. For example, increasing levels of isoflavonoidin soybean seeds would increase the efficiency of extraction and lowerthe cost of isoflavone-related products sold today for use in eitherreduction of serum cholesterol or in estrogen replacement therapy.Decreasing levels of isoflavonoid in soybean seeds would be beneficialfor production of soy-based infant formulas where the estrogenic effectsof isoflavonoid are undesirable. Raising levels of isoflavonoidphytoalexins in vegetative plant tissue could increase plant defenses topathogen attack, thereby improving plant disease resistance and loweringpesticide use rates. Manipulation of isoflavonoid levels in roots couldlead to improved nodulation and increased efficiencies of nitrogenfixation. To date, however, it has proven difficult to develop soybeanor other plant lines with consistently high levels of isoflavonoid.Because isoflavone synthase is the central reaction in pathwaysproducing isoflavonoids, identification of this functional gene isextremely important, and its manipulation via molecular techniques isexpected to allow production of soybeans and other plants with high,stable levels of isoflavonoid. Introduction of the isoflavone synthasegene in non-legume crop species including, but not limited to, corn,wheat, rice, sunflower, and canola could lead to synthesis ofisoflavonoids. The expression of isoflavonoids would confer to thesespecies disease resistance and/or properties which producehuman/livestock health benefits.

Substrates for isoflavone synthase may be limiting for synthesizing veryhigh levels of isoflavonoids in soybean, or for synthesizingisoflavonoids in non-legumes. It is desirable to increase the flux ofmetabolites through the phenylpropanoid pathway to provide additionalamounts of substrate to those occurring naturally. Different stressconditions such as UV irradiation, phosphate starvation, prolongedexposure to cold, and chemical (such as herbicide) treatment can causeactivation of the phenylpropanoid pathway. While these treatments mayproduce the desired substrate availability, it is more desirable to havea genetic means of activating the phenylpropanoid pathway. It is knownthat expression of genes encoding certain transcription factors canregulate the expression of various genes that encode enzymes of thephenylpropanoid pathway. These include, but are not limited to, the C1myb-type transcription factor of maize and the AmMyb305 of Antirrhinummajus. The C1 myb-type transcription factor of maize, in conjunctionwith the myc-type transcription factor R, activates chalcone synthaseand chalcone isomerase genes (Grotewold, E., et al. (1998) Plant Cell10:721-740). The Antirrhinum majus AmMyb305 activates the phenylalanineammonia lyase promoter (Sablowski, R. W., et al. (1994) EMBO J.13:128-137). Transcription factors such as these may be expressed inhost plant cells to activate expression of genes in the phenylpropanoidpathway thereby increasing the encoded enzyme activities and the flux ofcompounds through the pathway. Increases in the precursors to substratesof isoflavone synthase would enhance the production of isoflavonoids.

SUMMARY OF THE INVENTION

The instant invention relates to isolated nucleic acid sequencesencoding isoflavone synthase. In addition, this invention relates tonucleic acid sequences that are complementary to nucleic acid sequencesencoding isoflavone synthase. The nucleic acid sequences may be ofgenomic or cDNA origin and may contain introns.

In another embodiment, the instant invention relates to chimeric genesencoding isoflavone synthase or to chimeric genes that comprise nucleicacid sequences that are complementary to the nucleic acid sequencesencoding the enzyme, operably linked to suitable regulatory sequences,wherein expression of the chimeric genes results in production of levelsof isoflavone synthase in transformed host cells that are altered (i.e.,increased or decreased) from the levels produced in untransformed hostcells.

In a further embodiment, the instant invention concerns a transformedhost cell comprising in its genome a chimeric gene encoding anisoflavone synthase that is operably linked to suitable regulatorysequences. Expression of the chimeric gene results in production ofaltered levels of the enzyme in the transformed host cell. Thetransformed host cell can be of eukaryotic or prokaryotic origin, andincludes cells derived from higher plants and microorganisms. Theinvention also includes transformed plants that arise from transformedhost cells of higher plants, and seeds derived from such transformedplants.

An additional embodiment of the instant invention concerns a method ofaltering the level of expression of a plant isoflavone synthase in atransformed host cell comprising transforming a host cell with achimeric gene comprising a nucleic acid sequence (cDNA or genomic DNA)encoding an isoflavone synthase operably linked to suitable regulatorysequences and growing the transformed host cell under conditions thatare suitable for expression of the chimeric gene wherein expression ofthe chimeric gene results in production of altered levels of isoflavonesynthase in the transformed host cell. The altered levels of isoflavonesynthase may be higher due to overexpression, or may be lower due tocosuppression or anti sense suppression.

A further embodiment of the instant invention is a method for increasingthe amount of one or more isoflavonoids in a host cell. The methodcomprising the steps of transforming a host cell with a chimeric genecomprising a nucleic acid sequence encoding an isoflavone synthaseoperably linked to suitable regulatory sequences and growing thetransformed host cell under conditions that are suitable for expressionof the chimeric gene wherein expression of the chimeric gene results inproduction of an amount of isoflavonoids in the transformed host cellthat is greater than the amount of isoflavonoids that are produced in acell that is not transformed with the chimeric gene.

A further embodiment of the instant invention is a method for decreasingthe amount of one or more isoflavonoids in a host cell. The methodcomprising the steps of transforming a host cell with a chimeric genecomprising a nucleic acid sequence encoding all or a substantial portionof an isoflavone synthase operably linked to suitable regulatorysequences and growing the transformed host cell under conditions thatare suitable for expression of the chimeric gene wherein expression ofthe chimeric gene results in production of an amount of isoflavonoids inthe transformed host cell that is less than the amount of isoflavonoidsthat are produced in a cell that is not transformed with the chimericgene. The invention also includes transformed plants that arise fromtransformed host cells of higher plants, and seeds derived from suchtransformed plants.

An additional embodiment of the instant invention concerns a method forobtaining a nucleic acid sequence encoding all or substantially all ofan amino acid sequence encoding isoflavone synthase.

A still further embodiment of the instant invention concerns atransformed host cell comprising a chimeric gene encoding isoflavonesynthase and at least one chimeric gene encoding a transcription factorthat can regulate expression of one or more genes in the phenylpropanoidpathway. The invention also includes transformed plants that arise fromtransformed host cells of higher plants, and seeds derived from suchtransformed plants.

A further embodiment is a method of increasing the amount of one or moreisoflavonoids in a host cell comprising transforming a host cell with achimeric gene having a nucleic acid sequence encoding an isoflavonesynthase operably linked to suitable regulatory sequences and with atleast one chimeric gene having a nucleic acid sequence encoding atranscription factor that regulates expression of genes in thephenylpropanoid pathway, and growing the transformed host cell underconditions that are suitable for expression of the chimeric geneswherein expression of the chimeric genes result in production of anamount of one or more isoflavonoids in the transformed host cell that isgreater than the amount of the isoflavonoids that are produced in a cellthat is not transformed with the chimeric genes. The invention alsoincludes transformed plants that arise from transformed host cells ofhigher plants, and seeds derived from such transformed plants.

Yet a further embodiment of the present invention is a method ofaltering the level of isoflavonoids in a plant cell that is transformedwith a chimeric isoflavone synthase gene comprising exposing said cellto a phenylpropanoid pathway-altering agent. The phenylpropanoidpathway-altering agent may be a transcription factor or stress, forexample. Stress includes and is not limited to ultraviolet light,temperature, pressure, phosphate level, and herbicide treatment. Thetranscription factors may be a C1 myb-type transcription factor of maizeand a myc-type transcription factor R, or a chimera containing the maizeR region between the C1 DNA binding domain and the C1 activation domain.

BIOLOGICAL DEPOSIT

The following transformed yeast strain and vector plasmid have beendeposited with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20110-2209, and bears the followingdesignation, accession number and date of deposit. Yeast StrainAccession Number Date of Deposit Isoflavone Synthase GM1 ATCC 203606Jan. 27, 1999 Plasmid DP7951 ATCC PTA-371 Jul. 20, 1999

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing whichform a part of this application.

FIG. 1 depicts the phenylpropanoid metabolic pathway, and illustratesparticularly the biosynthesis of isoflavonoids.

FIGS. 2A and B presents the results of HPLC analyses of naringeninstandards. FIG. 2A presents the absorption spectra recorded at 260 nmand FIG. 2B presents the absorption spectra recorded at 280 nm.

FIGS. 3A and B presents the results of HPLC analyses of genisteinstandards. FIG. 3A presents the absorption spectra recorded at 260 nmand FIG. 3B presents the absorption spectra recorded at 280 nm.

FIGS. 4A and B presents the results of HPLC analyses of genistein andnaringenin from microsomes derived from elicitor-treated soybeanhypocotyls. Absorption spectra was recorded at 260 nm (FIG. 4A) and 280nm (FIG. 4B). Naringenin and genistein peaks are indicated.

FIGS. 5A and B presents the results of HPLC analyses of genistein andnaringenin from microsomes derived from non-treated soybean hypocotyls.Absorption spectra was recorded at 260 nm (FIG. 5A) and 280 nm (FIG.5B). Naringenin and genistein peaks are indicated.

FIGS. 6A and B presents the results of HPLC analyses of genistein andnaringenin from microsomes derived from elicitor-treated soybean cellsuspension cultures. Absorption spectra was recorded at 260 nm (FIG. 6A)and 280 nm (FIG. 6B). Naringenin and genistein peaks are indicated.

FIGS. 7A and B presents the results of HPLC analyses of genistein andnaringenin from microsomes derived from non-treated soybean cellsuspension cultures. Absorption spectra was recorded at 260 nm (FIG. 7A)and 280 nm (FIG. 7B). Naringenin peak is indicated.

FIGS. 8A and B presents the results of HPLC analyses of genistein andnaringenin in 75 μg of yeast microsomal proteins prior to incubation inthe presence of NADPH cofactor (negative control). Absorption spectrawas recorded at 260 nm (FIG. 8A) and 280 nm (FIG. 8B).

FIGS. 9A and B presents the results of HPLC analyses of genistein andnaringenin in 75 μg of yeast microsomal proteins after 1 h incubation inthe presence of NADPH cofactor. Absorption spectra was recorded at 260nm (FIG. 9A) and 280 nm (FIG. 9B).

FIGS. 10A and B presents the results of HPLC analyses of genistein andnaringenin in 75 μg of yeast microsomal proteins after 2 h incubation inthe presence of NADPH cofactor. Absorption spectra was recorded at 260nm (FIG. 10A) and 280 nm (FIG. 10B).

FIGS. 11A and B presents the results of HPLC analyses of genistein andnaringenin in 75 μg of yeast microsomal proteins after 3 h incubation inthe presence of NADPH cofactor. Absorption spectra was recorded at 260nm (FIG. 11A) and 280 nm (FIG. 11B).

FIG. 12 A and B presents the results of HPLC analyses of genistein andnaringenin in 75 μg of yeast microsomal proteins after 4 h incubation inthe presence of NADPH cofactor. Absorption spectra was recorded at 260nm (FIG. 12A) and 280 nm (FIG. 12B).

FIGS. 13A and B presents the results of HPLC analyses of genistein andnaringenin in 75 μg of yeast microsomal proteins after 14 h incubationin the presence of NADPH cofactor. Absorption spectra was recorded at260 nm (FIG. 13A) and 280 nm (FIG. 13B).

FIGS. 14A and B presents the results of HPLC analyses of genistein andnaringenin in 75 μg of yeast microsomal proteins after 40 minutesincubation in the presence of NADPH cofactor. Absorption spectra wasrecorded at 260 nm (FIG. 14A) and 280 nm (FIG. 14B).

FIGS. 15A and B presents the results of HPLC analyses of genistein andnaringenin in 150 μg of yeast microsomal proteins after 40 minutesincubation in the presence of NADPH cofactor. Absorption spectra wasrecorded at 260 nm (FIG. 15A) and 280 nm (FIG. 15B).

FIGS. 16A and B presents the results of HPLC analyses of genistein andnaringenin in 75 μg of yeast microsomal proteins after 4 h incubation inthe absence of NADPH cofactor. Absorption spectra was recorded at 260 nm(FIG. 16A) and 280 nm (FIG. 16B).

FIGS. 17A and B presents a comparison of the absorption spectra recordedby a diode array detector of a genistein standard (FIG. 17A; with anHPLC retention time of 3.128), and a reference spectrum (FIG. 17B).

FIGS. 18A and B presents a comparison of the absorption spectra recordedby a diode array detector of the newly synthesized peak located at theretention time of 3.131 in the HPLC analysis of yeast microsomesincubated for 14 h in the presence of NADPH on FIG. 18A and thereference spectrum on FIG. 18B.

FIGS. 19A, B, C, D and E presents the electropositive mass spectrumobtained for the peaks observed by HPLC analysis of yeast microsomesamples incubated with liquiritigenin. FIG. 19A corresponds to the peakat 273.2 m/z, FIG. 19B corresponds to the peak at 271 m/z, FIG. 19Ccorresponds to “peak 2”, FIG. 19D corresponds to liquiritigenin standard(the substrate), and FIG. 19E corresponds to daidzein standard (theproduct).

FIG. 20 depicts the plasmid map of pOY160.

FIG. 21 depicts the plasmid map of pOY206.

FIG. 22 depicts the plasmid map of pDP7951, having an ATCC accession No.PTA-371.

FIG. 23 depicts the plasmid map of pOY162.

FIG. 24 depicts the plasmid map of pKS93s.

FIG. 25 depicts the distribution of the isoflavonoid content of 25transgenic lines transformed with the isoflavone synthase sequence fromclone sgs1c.pk006.o20 and a control line. Bars represent the mean ofthree analyses for each line. The result of single factor ANOVA ispresented along with the least significant difference (LSD) at P≦0.01.The asterisk above the bars represents those lines with meanisoflavonoid concentrations significantly lower than control (bars 1through 6), or those lines with mean isoflavonoid concentrationssignificantly greater than control (bars 15 through 25) based on the LSDtest at P≦0.01.

FIG. 26 depicts the comparison of the rates of genistein and daidzeinsynthesis by microsomes of the yeast transformant GM 1. Samplesrepresenting incubation periods of 2, 4, 6, 8 and 10 h were analyzed byHPLC and the peak areas for genistein and daidzein were quantitated bycalibration with authentic genistein and daidzein standards. Assays wererepeated three times and the average amount of isoflavonoid synthesizedat each time point was plotted, with vertical lines representing errorbars.

FIG. 27 presents the results of HPLC analyses of daidzein andliquiritigenin in extracts from BMS cells before incubation in thepresence of NADPH cofactor (Panels A and B) and after 10 h incubation inthe presence of NADPH cofactor (Panels C and D). Absorption spectra wasrecorded at 260 nm (Panels A and C) and 280 nm (Panels B and D).

FIG. 28 depicts the plasmid map of pCW109-IFS.

The following sequence descriptions and Sequences Listing attachedhereto comply with the rules governing nucleotide and/or amino acidsequence disclosures in patent applications as set forth in 37 C.F.R.§1.821-1.825. The Sequence Listing contains the one letter code fornucleotide sequence characters and the three letter codes for aminoacids as defined in conformity with the IUPAC-IUB standards described inNucleic Acids Research 13:3021-3030 (1985) and in the BiochemicalJournal 219 (No. 2):345-373 (1984) which are herein incorporated byreference. The symbols and format used for nucleotide and amino acidsequence data comply with the rules set forth in 37 C.F.R. § 1.822.

SEQ ID NO:1 is the nucleotide sequence comprising the soybean cDNAinsert in clone sgs1c.pk006.o20 encoding an enzymatically activeisoflavone synthase.

SEQ ID NO:2 is the deduced amino acid sequence of an enzymaticallyactive soybean isoflavone synthase derived from the nucleotide sequenceof SEQ ID NO:1.

SEQ ID NO:3 is the nucleotide sequence of an oligonucleotide primer usedin the construction of yeast strain WHT1.

SEQ ID NO:4 is the nucleotide sequence of an oligonucleotide primer usedin the construction of the yeast strain WHT1.

SEQ ID NO:5 is the nucleotide sequence of an oligonucleotide primer usedto amplify the cDNA insert from clone sgs1c.pk006.o20.

SEQ ID NO:6 is the nucleotide sequence of an oligonucleotide primer usedto amplify the cDNA insert from clone sgs1c.pk006.o20.

SEQ ID NO:7 is the nucleotide sequence of an oligonucleotide primer usedfor PCR amplification of the soybean clone with sequence correspondingto the one found in NCBI General Identifier No. 2739005. Thisoligonucleotide sequence corresponds to nucleotides 3 to 26 of the NCBIsequence.

SEQ ID NO:8 is the nucleotide sequence of an oligonucleotide primer usedfor PCR amplification of the soybean clone with sequence correspondingto the one found in NCBI General Identifier No. 2739005. Thisoligonucleotide sequence corresponds to the complement of nucleotides1798 to 1824 of the NCBI sequence.

SEQ ID NO:9 is the nucleotide sequence of an enzymatically activesoybean isoflavone synthase having an NCBI General Identifier No.2739005.

SEQ ID NO:10 is the deduced amino acid sequence of an enzymaticallyactive soybean isoflavone synthase derived from of SEQ ID NO:9 andhaving an NCBI General Identifier No. 2739006.

SEQ ID NO:11 is the nucleotide sequence of an oligonucleotide primerused for PCR amplification of the isoflavone synthase genes from mungbean, red clover, white clover, lentil, hairy vetch, alfalfa, lupine andsnow pea.

SEQ ID NO:12 is the nucleotide sequence of an oligonucleotide primerused for PCR amplification of the isoflavone synthase genes from mungbean, red clover, white clover, lentil, hairy vetch, alfalfa, lupine andsnow pea.

SEQ ID NO:13 is the nucleotide sequence of an oligonucleotide primerused in the second round of PCR amplification of the white clover,lentil, hairy vetch, alfalfa and lupine isoflavone synthase genes.

SEQ ID NO:14 is the nucleotide sequence of an oligonucleotide primerused in the second round of PCR amplification of the white clover,lentil, hairy vetch, alfalfa and lupine isoflavone synthase genes.

SEQ ID NO:15 is the nucleotide sequence comprising the alfalfa cDNAinsert in clone alfalfa1 encoding an almost entire alfalfa isoflavonesynthase.

SEQ ID NO:16 is the deduced amino acid sequence of an almost entirealfalfa isoflavone synthase derived from the nucleotide sequence of SEQID NO:15.

SEQ ID NO:17 is the nucleotide sequence comprising the hairy vetch cDNAinsert in clone hairy vetch1 encoding an almost entire hairy vetchisoflavone synthase.

SEQ ID NO:18 is the deduced amino acid sequence of an almost entirehairy vetch isoflavone synthase derived from the nucleotide sequence ofSEQ ID NO:17.

SEQ ID NO:19 is the nucleotide sequence comprising the lentil cDNAinsert in clone lentil1 encoding an almost entire lentil isoflavonesynthase.

SEQ ID NO:20 is the deduced amino acid sequence of an almost entirelentil isoflavone synthase derived from the nucleotide sequence of SEQID NO:19.

SEQ ID NO:21 is the nucleotide sequence comprising the lentil cDNAinsert in clone lentil2 encoding an almost entire lentil isoflavonesynthase.

SEQ ID NO:22 is the deduced amino acid sequence of an almost entirelentil isoflavone synthase derived from the nucleotide sequence of SEQID NO:21.

SEQ ID NO:23 is the nucleotide sequence comprising the mung bean cDNAinsert in clone mung bean1 encoding an entire mung bean isoflavonesynthase.

SEQ ID NO:24 is the deduced amino acid sequence of an entire mung beanisoflavone synthase derived from SEQ ID NO:23.

SEQ ID NO:25 is the nucleotide sequence comprising the mung bean cDNAinsert in clone mung bean2 encoding an entire mung bean isoflavonesynthase.

SEQ ID NO:26 is the deduced amino acid sequence of an entire mung beanisoflavone synthase derived from SEQ ID NO:25.

SEQ ID NO:27 is the nucleotide sequence comprising the mung bean cDNAinsert in clone mung bean3 encoding an entire mung bean isoflavonesynthase.

SEQ ID NO:28 is the deduced amino acid sequence of an entire mung beanisoflavone synthase derived from SEQ ID NO:27.

SEQ ID NO:29 is the nucleotide sequence comprising the mung bean cDNAinsert in clone mung bean4 encoding an entire mung bean isoflavonesynthase.

SEQ ID NO:30 is the deduced amino acid sequence of an entire mung beanisoflavone synthase derived from SEQ ID NO:30.

SEQ ID NO:31 is the nucleotide sequence comprising the red clover cDNAinsert in clone red clover1 encoding an entire red clover isoflavonesynthase.

SEQ ID NO:32 is the deduced amino acid sequence of an entire red cloverisoflavone synthase derived from SEQ ID NO:31.

SEQ ID NO:33 is the nucleotide sequence comprising the red clover cDNAinsert in clone red clover2 encoding an entire red clover isoflavonesynthase.

SEQ ID NO:34 is the deduced amino acid sequence of an entire red cloverisoflavone synthase derived from SEQ ID NO:33.

SEQ ID NO:35 is the nucleotide sequence comprising the snow pea cDNAinsert in clone snow peal encoding an entire snow pea isoflavonesynthase.

SEQ ID NO:36 is the deduced amino acid sequence of an entire snow peaisoflavone synthase derived from SEQ ID NO:37.

SEQ ID NO:37 is the nucleotide sequence comprising the white clover cDNAinsert in clone white clover1 encoding an almost entire white cloverisoflavone synthase.

SEQ ID NO:38 is the deduced amino acid sequence of an almost entirewhite clover isoflavone synthase derived from SEQ ID NO:37.

SEQ ID NO:39 is the nucleotide sequence comprising the white clover cDNAinsert in clone white clover2 encoding an almost entire white cloverisoflavone synthase.

SEQ ID NO:40 is the deduced amino acid sequence of an almost entirewhite clover isoflavone synthase derived from SEQ ID NO:39.

SEQ ID NO:41 is the nucleotide sequence of an oligonucleotide primerused for PCR amplification of the isoflavone synthase coding region inclone sgs1c.pk006.o20. SEQ ID NO:42 is the nucleotide sequence of anoligonucleotide primer used for PCR amplification of the isoflavonesynthase coding region in clone sgs1c.pk006.o20.

SEQ ID NO:43 is the nucleotide sequence of an oligonucleotide primerused to determine the transcription of the soybean isoflavone synthasein transgenic tobacco.

SEQ ID NO:44 is the nucleotide sequence of an oligonucleotide primerused to determine the transcription of the soybean isoflavone synthasein transgenic tobacco.

SEQ ID NO:45 is the nucleotide sequence of an oligonucleotide primer tothe maize R coding region used to amplify genomic DNA to determine thepresence of a chimera containing the maize R region between the regionencoding the C1 DNA binding domain and the C1 activation domain (CRC) intransgenic corn cells.

SEQ ID NO:46 is the nucleotide sequence of an oligonucleotide primer tothe 3′ untranslated region from potato protease inhibitor II gene usedto amplify genomic DNA to determine the presence of CRC in transgeniccorn cells.

SEQ ID NO:47 is the nucleotide sequence comprising the sugarbeet cDNAinsert in clone sugarbeet1, encoding an almost entire sugarbeetisoflavone synthase.

SEQ ID NO:48 is the deduced amino acid sequence of an almost entiresugarbeet isoflavone synthase derived from SEQ ID NO:47.

SEQ ID NO:49 is the nucleotide sequence of an oligonucleotide primerused for the PCR amplification of the soybean isoflavone synthase codingregion in clone sgs1c.pk006.o20.

SEQ ID NO:50 is the nucleotide sequence of an oligonucleotide primerused for the PCR amplification of the soybean isoflavone synthase codingregion in clone sgs1c.pk006.o20.

SEQ ID NO:51 is the nucleotide sequence of an oligonucleotide primerused to amplify the genomic sequence comprising the isoflavone synthasein clone sgs1c.pk006.o20.

SEQ ID NO:52 is the nucleotide sequence of a genomic fragment encodingthe isoflavone synthase in clone sgs1c.pk006.o20.

SEQ ID NO:53 is the nucleotide sequence of a genomic fragment encodingthe CYP93C1 isoflavone synthase.

SEQ ID NO:54 is the nucleotide sequence comprising the lupine cDNAinsert in clone lupine1 encoding an entire lupine isoflavone synthase.

SEQ ID NO:55 is the deduced amino acid sequence of an entire lupineisoflavone synthase derived from SEQ ID NO:54.

SEQ ID NO:56 is the nucleotide sequence comprising the alfalfa cDNAinsert in clone alfalfa2 encoding an almost entire alfalfa isoflavonesynthase.

SEQ ID NO:57 is the amino acid sequence of an almost entire alfalfaisoflavone synthase derived from SEQ ID NO:56.

SEQ ID NO:58 is the nucleotide sequence comprising the alfalfa cDNAinsert in clone alfalfa3 encoding an almost entire alfalfa isoflavonesynthase.

SEQ ID NO:59 is the amino acid sequence of an almost entire alfalfaisoflavone synthase derived from SEQ ID NO:58.

SEQ ID NO:60 is the amino acid sequence comprising the sugarbeet cDNAinsert in clone sugarbeet2, encoding an almost entire sugarbeetisoflavone synthase.

SEQ ID NO:61 is the deduced amino acid sequence of an almost entiresugarbeet isoflavone synthase derived from SEQ ID NO:60.

SEQ ID NO:62 is the nucleotide sequence of an oligonucleotide primerused for the PCR amplification of the soybean chalcone reductase codingregion in clone src3c.pk009.e4.

SEQ ID NO:63 is the nucleotide sequence of an oligonucleotide primerused for the PCR amplification of the soybean chalcone reductase codingregion in clone src3c.pk009.e4.

SEQ ID NO:64 is the nucleotide sequence of an oligonucleotide primerused for the PCR amplification of the soybean chalcone reductase presentin monocot cells.

SEQ ID NO:65 is the nucleotide sequence of an oligonucleotide primerused for the PCR amplification of the soybean chalcone reductase presentin monocot cells.

SEQ ID NO:66 is the amino acid sequence of the consensus sequenceproduced by the Megalign Program using the Clustal method and the aminoacid sequences depicted in SEQ ID NOs:2, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 48, 55, 57, 59, and 61.

DETAILED DESCRIPTION OF THE INVENTION

The instant invention discloses nucleotide and amino acid sequences forisoflavone synthases from legumes such as soybean, alfalfa, lupine,hairy vetch, lentil, mung bean, red clover, snow pea, and white cloverand non-legumes such as sugarbeet. As the enzyme that catalyzes thefirst step of the isoflavonoid branch of the phenylpropanoid pathway(see FIG. 1), altering the level of this enzyme may be useful forchanging isoflavonoid content.

Plant P450 enzymes catalyze a diverse range of reactions, includingmolecular transformations in primary metabolism, and in the metabolismand detoxification of xenobiotics. Although tentative identification ofany given gene or conceptual translation product as a P450 is relativelysimple based on its similarity to other known P450s, the assignment ofactual catalytic function cannot necessarily be inferred from nucleicacid or protein sequence information alone. The instant disclosuredemonstrates and teaches the identification of a cDNA from soybean thatencodes isoflavone synthase based on the ability of the encodedpolypeptide to convert the normal substrate for the reaction,2S-flavanone, to genistein. Demonstration of activity has beenaccomplished in subcellular fractions of a yeast strain, WHT1, which hasbeen specifically altered to also express a P450 reductase fromHelianthus tuberosum. In this manner, and using the materials identifiedand described herein, other nucleic acid sequences from soybean and fromother plants that are predicted to encode P450s may be tested todetermine whether any of those P450's possess isoflavone synthaseactivity.

“The isoflavonoids are biogeneticaly related to the flavonoids butconstitute a distinctly separate class in that they contain a rearrangedC15 skeleton and may be regarded as derivatives of 3-phenylchroman.”Isoflavonoids. Dewick, P. M. (1982) in The Flavonoids: Advances inResearch, Harborne, J. B. and Mabry, T. J., Ed., pp 535-640, Chapman andHall Ltd, New York. Oxidative rearrangement of a flavanone precursorwith a 2,3-aryl shift yields an isoflavonoid. Isoflavones are the mostabundant of the natural isoflavonoid derivatives, with over 160isoflavone aglycones being recognized.

In the context of this disclosure, a number of terms shall be utilized.As used herein, a “nucleic acid sequence” is a polymer of RNA or DNAthat is single- or double-stranded, optionally containing synthetic,non-natural or altered nucleotide bases. A nucleic acid sequence in theform of a polymer of DNA may be comprised of one or more segments ofcDNA, genomic DNA or synthetic DNA.

As used herein, “substantially similar” refers to nucleic acid sequenceswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the polypeptide encoded by the nucleotide sequence. “Substantiallysimilar” also refers to nucleic acid sequences wherein changes in one ormore nucleotide bases does not affect the ability of the nucleic acidsequence to mediate alteration of gene expression by gene silencingthrough for example antisense or co-suppression technology.“Substantially similar” also refers to modifications of the nucleic acidfragments of the instant invention such as deletion or insertion of oneor more nucleotides that do not substantially affect the functionalproperties of the resulting transcript vis-à-vis the ability to mediategene silencing or alteration of the functional properties of theresulting protein molecule. It is therefore understood that theinvention encompasses more than the specific exemplary nucleotide oramino acid sequences and includes functional equivalents thereof.

For example, it is well known in the art that antisense suppression andco-suppression of gene expression may be accomplished using nucleic acidfragments representing less than the entire coding region of a gene, andby nucleic acid fragments that do not share 100% sequence identity withthe gene to be suppressed. Moreover, alterations in a nucleic acidsequence which result in the production of a chemically equivalent aminoacid at a given site, but do not effect the functional properties of theencoded polypeptide, are well known in the art. Thus, a codon for theamino acid alanine, a hydrophobic amino acid, may be substituted by acodon encoding another less hydrophobic residue, such as glycine, or amore hydrophobic residue, such as valine, leucine, or isoleucine.Similarly, changes which result in substitution of one negativelycharged residue for another, such as aspartic acid for glutamic acid, orone positively charged residue for another, such as lysine for arginine,can also be expected to produce a functionally equivalent product.Nucleotide changes which result in alteration of the N-terminal andC-terminal portions of the polypeptide molecule would also not beexpected to alter the activity of the polypeptide. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity of the encodedproducts.

Moreover, substantially similar nucleic acid sequences may also becharacterized by their ability to hybridize. Estimates of such homologyare provided by either DNA-DNA or DNA-RNA hybridization under conditionsof stringency as is well understood by those skilled in the art (Hamesand Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford,U.K.). Stringency conditions can be adjusted to screen for moderatelysimilar sequences, such as homologous sequences from distantly relatedorganisms, to highly similar sequences, such as genes that duplicatefunctional enzymes from closely related organisms. Post-hybridizationwashes determine stringency conditions. One set of preferred conditionsuses a series of washes starting with 6×SSC, 0.5% SDS at roomtemperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30min. A more preferred set of stringent conditions uses highertemperatures in which the washes are identical to those above except forthe temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS wasincreased to 60° C. Another preferred set of highly stringent conditionsuses two final washes in 0.1×SSC, 0.1% SDS at 65° C.

Substantially similar nucleic acid sequences of the instant inventionmay also be characterized by their percent identity to the nucleic acidsequences disclosed herein, as determined by algorithms commonlyemployed by those skilled in this art. Preferred are those nucleic acidsequences whose sequences are at least about 85% identical and morepreferably at least about 90% identical to the nucleotide sequencesreported herein. More preferred are nucleic acid sequences that are atleast about 90% identical and more preferably at least about 95%identical to the nucleotide sequences reported herein. More preferredare nucleic acid sequences that are 95% identical to the nucleotidesequences reported herein. Sequence alignments and percent identitycalculations were performed using the Megalign program of the LASARGENEbioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiplealignment of the sequences was performed using the Clustal method ofalignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the defaultparameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parametersfor pairwise alignments using the Clustal method were KTUPLE 2, GAPPENALTY=5, WINDOW=4 and DIAGONALS SAVED=4.

Substantially similar nucleic acid sequences of the instant inventionmay also be characterized by the percent identity of the amino acidsequences that they encode to the amino acid sequences disclosed herein,as determined by algorithms commonly employed by those skilled in thisart. Preferred are those nucleic acid sequences whose nucleotidesequences encode amino acid sequences that are at least about 95%identical and even more preferably at least about 98% identical to theamino acid sequences reported herein. Sequence alignments and percentidentity calculations were performed using the Megalign program of theLASARGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).Multiple alignment of the sequences was performed using the Clustalmethod of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) withthe default parameters (GAP PENALTY-=10, GAP LENGTH PENALTY=10). Defaultparameters for pairwise alignments using the Clustal method were KTUPLE1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more contiguous nucleotides isnecessary in order to putatively identify a polypeptide or nucleic acidsequence as homologous to a known protein or gene. Moreover, withrespect to nucleotide sequences, gene-specific oligonucleotide probescomprising 30 or more contiguous nucleotides may be used insequence-dependent methods of gene identification (e.g., Southernhybridization) and isolation (e.g., in situ hybridization of bacterialcolonies or bacteriophage plaques). In addition, short oligonucleotidesof 12 or more nucleotides may be used as amplification primers in PCR inorder to obtain a particular nucleic acid sequence comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises a nucleotide sequence that will afford specific identificationand/or isolation of a nucleic acid sequence comprising the sequence. Theinstant specification teaches amino acid and nucleotide sequencesencoding polypeptides that comprise one or more particular plantproteins. The skilled artisan, having the benefit of the sequences asreported herein, may now use all or a substantial portion of thedisclosed sequences for purposes known to those skilled in this art.Accordingly, the instant invention comprises the complete sequences asreported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid sequence comprising a nucleotide sequencethat encodes all or a substantial portion of the amino acid sequencesset forth herein. The skilled artisan is well aware of the “codon-bias”exhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. Therefore, when synthesizing a nucleic acidsequence for improved expression in a host cell, it is desirable todesign the nucleic acid fragment such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

“Synthetic nucleic acid fragments” can be assembled from oligonucleotidebuilding blocks that are chemically synthesized using procedures knownto those skilled in the art. These building blocks are ligated andannealed to form larger nucleic acid sequences which may then beenzymatically assembled to construct the entire desired nucleic acidsequence. “Chemically synthesized”, as related to nucleic acid sequence,means that the component nucleotides were assembled in vitro. Manualchemical synthesis of nucleic acid sequences may be accomplished usingwell established procedures, or automated chemical synthesis can beperformed using one of a number of commercially available machines.Accordingly, the nucleic acid sequences can be tailored for optimal geneexpression based on optimization of nucleotide sequence to reflect thecodon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

“Gene” refers to a nucleic acid sequence that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Coding sequence” refers to a nucleotide sequence that codes for aspecific amino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is anucleotide sequence which can stimulate promoter activity. It may be aninnate element of the promoter or a heterologous element inserted toenhance the level and/or tissue-specificity of a promoter. Promoters maybe derived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven comprise synthetic nucleotide segments. It is understood by thoseskilled in the art that different promoters may direct the expression ofa gene in different tissues or cell types, or at different stages ofdevelopment, or in response to different environmental conditions.Promoters which cause a nucleic acid sequence to be expressed in mostcell types at most times are commonly referred to as “constitutivepromoters”. “Organ-specific” or “development-specific” promoters arethose that direct gene expression almost exclusively in specific organs,such as leaves or seeds, or at specific development stages in an organ,such as in early or late embryogenesis, respectively. New promoters ofvarious types useful in plant cells are constantly being discovered;numerous examples may be found in the compilation by Okamuro andGoldberg (1989) Biochemistry of Plants 15:1-82. It is further recognizedthat since in most cases the exact boundaries of regulatory sequenceshave not been completely defined, nucleic acid sequences of differentlengths may have identical promoter activity.

The expression of foreign genes in plants is well established (De Blaereet al. (1987) Meth. Enzymol. 143:277-291). Proper level of expression ofmRNAs may require the use of different chimeric genes utilizingdifferent promoters. Such chimeric genes can be transferred into hostplants either together in a single expression vector or sequentiallyusing more than one vector. Expression in plants will use regulatorysequences functional in such plants.

The origin of the promoter chosen to drive the expression of the codingsequence is not critical as long as it has sufficient transcriptionalactivity to accomplish the invention by expressing translatable mRNA forthe desired protein genes in the desired host tissue.

The “translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) MolecularBiotechnology 3:225-236).

The “3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1:671-680.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptide by the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to an RNAtranscript that includes the mRNA and so can be translated into apolypeptide by the cell. “Antisense RNA” refers to an RNA transcriptthat is complementary to all or part of a target primary transcript ormRNA and that blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

The term “operably linked” refers to the association of two or morenucleic acid sequences on a single nucleic acid sequence so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid sequence of the invention. Expression may also refer totranslation of mRNA into a polypeptide. “Antisense inhibition” refers tothe production of antisense RNA transcripts capable of suppressing theexpression of the target protein. “Overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms. “Co-suppression”refers to the production of sense RNA transcripts capable of suppressingthe expression of identical or substantially similar foreign orendogenous genes (U.S. Pat. No. 5,231,020, incorporated herein byreference).

“Altered levels” refers to the production of gene product(s) intransgenic organisms in amounts or proportions that differ from that ofnormal or non-transformed organisms.

“Transformation” refers to the transfer of a nucleic acid sequence intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. (1987) Meth. Enzymol 143:277) and particle-acceleratedor “gene gun” transformation technology (Klein et al. (1987) Nature(London) 327:70-73; U.S. Pat. No. 4,945,050, incorporated herein byreference).

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook etal. Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory Press: Cold Spring Harbor, 1989 (hereinafter “Sambrook”).

A nucleic acid sequence encoding a soybean isoflavone synthase wasisolated and identified from a cDNA library. Nucleic acid sequencesencoding three alfalfa, one hairy vetch, one snow pea, one lupine, twolentil, two red clover, two white clover, two sugarbeet, and four mungbean isoflavone synthases have been isolated-using RT-PCR. Nucleic acidsequences encoding two soybean isoflavone synthases have been isolatedfrom genomic DNA. The nucleic acid sequences of the instant inventionmay be used to isolate cDNAs and genes encoding homologous enzymes fromthe same or other plant species. Isolation of homologous genes usingsequence-dependent protocols is well known in the art. Examples ofsequence-dependent protocols include, but are not limited to, methods ofnucleic acid hybridization, and methods of DNA and RNA amplification asexemplified by various uses of nucleic acid amplification technologies(e.g., polymerase chain reaction, ligase chain reaction).

For example, genes encoding other isoflavone synthase proteins, eitheras cDNAs or genomic DNAs, could be isolated directly by using all or aportion of the instant nucleic acid sequence as aDNA hybridization probeto screen libraries from any desired plant employing methodology wellknown to those skilled in the art. Specific oligonucleotide probes basedupon the instant nucleic acid sequence can be designed and synthesizedby methods known in the art (Sambrook). Moreover, the entire sequencecan be used directly to synthesize DNA probes by methods known to theskilled artisan such as random primers DNA labeling, nick translation,or end-labeling techniques, or RNA probes using available in vitrotranscription systems. In addition, specific primers can be designed andused to amplify a part of or full-length of the instant sequences. Theresulting amplification products can be labeled directly duringamplification reactions or labeled after amplification reactions, andused as probes to isolate full-length cDNA or genomic fragments underconditions of appropriate stringency.

In addition, two short segments of the instant nucleic acid sequencesmay be used in polymerase chain reaction protocols to amplify longernucleic acid sequences encoding homologous genes from DNA or RNA. Thepolymerase chain reaction may also be performed on a library of clonednucleic acid sequences wherein the sequence of one primer is derivedfrom the instant nucleic acid sequences, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor encoding plant genes. Alternatively,the second primer sequence may be based upon sequences derived from thecloning vector. For example, the skilled artisan can follow the RACEprotocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998-9002)to generate cDNAs by using PCR to amplify copies of the region between asingle point in the transcript and the 3′ or 5′ end. Primers oriented inthe 3′ and 5′ directions can be designed from the instant sequences.Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific3′ or 5′ cDNA sequences can be isolated (Ohara et al. (1989) Proc. Natl.Acad. Sci. USA 86:5673-5677; Loh et al. (1989) Science 243:217-220).Products generated by the 3′ and 5′ RACE procedures can be combined togenerate full-length cDNAs (Frohman and Martin (1989) Techniques 1:165).

Availability of the instant nucleotide and deduced amino acid sequencesfacilitates immunological screening of cDNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequences may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can be then be used to screen cDNA expression libraries toisolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol.36:1; Sambrook).

The nucleic acid sequence of the instant invention may be used to createtransgenic plants and transgenic seeds in which expression of nucleicacid sequences (or their complements) encoding the disclosed enzymeresult in levels of the corresponding endogenous enzyme that are higheror lower than normal. Alternatively, expression of the instant nucleicacid sequence may result in the production of the encoded enzyme in celltypes or developmental stages in which they are not normally found.Either strategy would have the effect of altering the level ofisoflavonoids.

For example, overexpression of isoflavone synthase may result in anincrease in isoflavonoid content in legumes. Increased isoflavonoidcontent in legumes has been shown to be associated with beneficialhealth effects in humans. In contrast, certain soy food products wouldbenefit from lower levels of isoflavonoid due to adverse effects onflavor.

Overexpression of the proteins of the instant invention may beaccomplished by first constructing a chimeric gene in which the codingregion is operably linked to a promoter capable of directing expressionof a gene in the desired tissues at the desired stage of development.The chimeric gene may comprise promoter sequences and translation leadersequences derived from the same genes. 3′ Non-coding sequences encodingtranscription termination signals may also be provided. The instantchimeric gene may also comprise one or more introns in order tofacilitate gene expression.

Plasmid vectors comprising the isolated polynucleotide (or chimericgene) may be constructed. The choice of plasmid vector is dependent uponthe method that will be used to transform host plants. The skilledartisan is well aware of the genetic elements that must be present onthe plasmid vector in order to successfully transform, select andpropagate host cells containing the chimeric gene. The skilled artisanwill also recognize that different independent transformation eventswill result in different levels and patterns of expression (Jones et al.(1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics218:78-86), and thus that multiple events must be screened in order toobtain lines displaying the desired expression level and pattern. Suchscreening may be accomplished by Southern analysis of DNA, Northernanalysis of mRNA expression, Western analysis of protein expression, orphenotypic analysis.

The nucleic acid sequence of the instant invention may be used to createtransgenic plants that have increased expression of the disclosed enzymeand that are additionally transformed with a chimeric gene encoding atranscription factor that regulates expression of one or more genes inthe phenylpropanoid pathway. The chimeric transcription factor gene hasregulatory sequences such that its expression is coordinated with thatof the isoflavone synthase gene developmentally and preferably withinthe same cell type. This combination of expression of isoflavonesynthase and transcription factor regulating phenylpropanoid pathwaygenes has the effect of enhancing the level of isoflavonoid synthesisdue to increased levels of substrates for isoflavone synthase. Thechimeric transcription factor gene regulates expression of at least onegene in the phenylpropanoid pathway. While not intending to be bound byany theory or theories of operation it is believed to regulate as manyas two, three or four genes in the phenylpropanoid pathway.

For example, a plant cell that does not naturally produce isoflavonoidsand does not have an active phenylpropanoid pathway would not producethe substrates for isoflavone synthase to convert to isoflavonoids.Activation of the phenylpropanoid pathway in the desired cells or at thedesired developmental stage would provide these substrates allowing thesynthesis of isoflavonoids.

The present invention is also directed to a method of altering the levelof isoflavonoids in a cell comprising exposing said cell to aphenylpropanoid pathway altering agent. The cell may be a plant cellsuch as a monocot, including and not limited to corn, or a dicot, suchas soybean, for example. A phenylpropanoid pathway altering agent may beany agent that results in an increase or decrease in the level ofexpression of an enzyme in the phenylpropanoid pathway, such asisoflavone synthase, phenylalanine ammonia lyase, chalcone synthase,among others. Such phenylpropanoid pathway altering agents include andare not limited to a transcription factor and stress. Transcriptionfactors include and are not limited to chimeric transcription factors, achimera containing the maize R region between the region encoding the C1DNA binding domain and the C1 activation domain (CRC) for example.Stresses to a plant cell include ultraviolet light, temperature,pressure, chemicals including and not limited to herbicides, andphosphate level. Phosphate levels may be increased or decreased suchthat decreasing phosphate levels may result in phosphate starvation.

It may also be desirable to reduce or eliminate expression of genesencoding the instant polypeptides in plants for some applications. Inorder to accomplish this, a chimeric gene designed for co-suppression ofthe instant polypeptide can be constructed by linking a gene or genesequence encoding that polypeptide to plant promoter sequences.Alternatively, a chimeric gene designed to express antisense RNA for allor part of the instant nucleic acid sequence can be constructed bylinking the gene or gene sequence in reverse orientation to plantpromoter sequences. Either the co-suppression or antisense chimericgenes could be introduced into plants via transformation whereinexpression of the corresponding endogenous genes are reduced oreliminated.

Molecular genetic solutions to the generation of plants with alteredgene expression have a decided advantage over more traditional plantbreeding approaches. Changes in plant phenotypes can be produced byspecifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression ofspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

The person skilled in the art will know that special considerations areassociated with the use of antisense or cosuppresion technologies inorder to reduce expression of particular genes. For example, the properlevel of expression of sense or antisense genes may require the use ofdifferent chimeric genes utilizing different regulatory elements knownto the skilled artisan. Once transgenic plants are obtained by one ofthe methods described above, it will be necessary to screen individualtransgenics for those that most effectively display the desiredphenotype. Accordingly, the skilled artisan will develop methods forscreening large numbers of transformants. The nature of these screenswill generally be chosen on practical grounds. For example, one canscreen by looking for changes in gene expression by using antibodiesspecific for the protein encoded by the gene being suppressed, or onecould establish assays that specifically measure enzyme activity. Apreferred method will be one which allows large numbers of samples to beprocessed rapidly, since it will be expected that a large number oftransformants will be negative for the desired phenotype.

The instant isoflavone synthases (or portions of the enzymes) may beproduced in heterologous host cells, particularly in the cells ofmicrobial hosts, and can be used to prepare antibodies to the enzymes bymethods well known to those skilled in the art. The antibodies areuseful for detecting the enzymes in situ in cells or in vitro in cellextracts. Preferred heterologous host cells for production of isoflavonesynthase are yeast hosts. Yeast expression systems and expressionvectors containing regulatory sequences that direct high levelexpression of foreign proteins are well known to those skilled in theart. Any of these could be used to construct chimeric genes forproduction of the instant isoflavone synthase. These chimeric genescould then be introduced into appropriate hosts via transformation toprovide high level expression of the enzymes. An example of a vector forhigh level expression of the instant isoflavone synthase in a yeast hostis provided (Example 5).

All or a substantial portion of the nucleic acid sequences of theinstant invention may also be used as probes for genetically andphysically mapping the genes that they are a part of, and as markers fortraits linked to those genes. Such information may be useful in plantbreeding in order to develop lines with desired phenotypes. For example,the instant nucleic acid sequences may be used as restriction sequencelength polymorphism (RFLP) markers. Southern blots (Maniatis) ofrestriction-digested plant genomic DNA may be probed with the nucleicacid sequences of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1: 174-181) in order toconstruct a genetic map. In addition, the nucleic acid sequences of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet.32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bematzky and Tanksley (1986) Plant Mol. Biol.Reporter 4(1):37-41. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences mayalso be used for physical mapping (i.e., placement of sequences onphysical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: APractical Guide, Academic press 1996, pp. 319-346, and references citedtherein).

In another embodiment, nucleic acid probes derived from the instantnucleic acid sequences may be used in direct fluorescence in situhybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154).Although current methods of FISH mapping favor use of large clones(several to several hundred KB; see Laan et al. (1995) Genome Research5:13-20), improvements in sensitivity may allow performance of FISHmapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic andphysical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J Lab. Clin. Med 114(2):95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997)Nature Genetics 7:22-28) and Happy Mapping (Dear and Cook (1989) NucleicAcid Res. 17:6795-6807). For these methods, the sequence of a nucleicacid fragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

The physiological activities associated with isoflavonoids in bothplants and humans makes the manipulation of their contents in cropplants highly desirable. For example, increasing levels of isoflavonoidsin soybean seeds would increase the efficiency of extraction and lowerthe cost of isoflavonoid-related products sold. Decreasing levels ofisoflavonoids in soybean seeds would be beneficial for production ofsoy-based infant formulas where the estrogenic effects of isoflavonoidsare undesirable. Decreasing levels of isoflavonoids may also increasepalatability of soy foods. Raising levels of isoflavonoid phytoalexinsin vegetative plant tissue could increase plant defenses to pathogenattack, thereby improving resistance and lowering the need for pesticideuse. Manipulation of isoflavonoid levels in roots could lead to improvednodulation and increased efficiencies of nitrogen fixation. To date,however, it has proven difficult to develop soybean or other plant lineswith consistently high levels of isoflavonoids.

Identification of the functional isoflavone synthase gene is extremelyimportant because isoflavone synthase catalyzes the central reaction inpathways producing isoflavonoids. Manipulation of the isoflavonesynthase gene via molecular techniques is expected to allow productionof soybeans and other plants with high, stable levels of isoflavonoids.Introduction of the isoflavone synthase gene in non-legume crop speciesincluding, but not limited to, corn, wheat, rice, sunflower, and canolacould lead to synthesis of isoflavonoids in these species. Synthesis ofisoflavonoids would 1) confer disease resistance to the crops and/or 2)produce crops which would benefit human and/or livestock health.

EXAMPLES

The present invention is further defined in the following Examples, inwhich all parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions.

Example 1 Microsome Preparation from Elicitor-Treated Soybean Hypocotylsand Elicitor-Treated Cell Suspension Culture

Elicitor Treatment of Soybean Seeds

Soybean seeds were placed on a bed of vermiculite (5 to 6 cm thick) andcovered with a layer of vermiculite about 2 cm thick. Seeds weregerminated for five days in a growth chamber until the average length ofhypocotyls reached to about 3 to 4 cm. The growth chamber was kept at acycle that consisted of a 14 h light period at 25° C. and a 10 h darkperiod at 21° C. Illumination was supplied from cool white fluorescentand incandescent lamps that provide a photon flux density of 450μEm⁻²s⁻¹. Soybean hypocotyls were pulled out from the vermiculite bedand were placed on wet paper towels. The soybean hypocotyls were dividedinto two groups: one of the groups was treated with elicitor and theother was not treated.

Elicitor treatment was conducted as follows. The epidermal surfaces ofthe hypocotyls were opened using a razor blade. The incisions wereapproximately 2 cm long and 1 to 2 mm deep; one was made on eachhypocotyl. Fungal-derived elictors were prepared by the method of Sharpet al. (Sharp, J. K. et al. (1984) J. Biol. Chem. 259:11312-11320).Twenty micrograms of acidified fungal elicitors were dissolved in 20 μLof 10 mM KH₂PO₄, and were then applied to the wound of a hypocotyl Thetreated hypocotyls were incubated for 15 h in the dark at roomtemperature and 100% humidity. At the end of the incubation period, thehypocotyls were sectioned closely below the cotyledonal node and wereimmediately frozen in liquid nitrogen and stored at −76° C. until used.Non-elicitor-treated hypocotyls were handled in the same manner as wereelicitor-treated hypocotyls, except for wounding and elicitorapplication. The non-treated hypocotyls were used as a negative controlof isoflavone synthase induction.

Elicitor Treatment of Soybean Cell Suspension Culture

Soybean suspension cell cultures were grown at 25° C. in 250 mL flasksthat were tightly covered with two layers of aluminum foil to preventillumination. Cells were grown in 35 mL of Murashige and Skoog medium(Gibco BRL) supplemented with 0.75 mg/L 2,4-dichlorophenoxyacetic acidand 0.55 mg/mL 6-benzyl aminopurine. Cells were diluted (1:3 ratio) intofresh medium every 7 days and elicitor treatment was conducted 3 daysafter cell dilution. One hundred fifty milligrams of the same fungalelicitor used to treat the hypocotyls was dissolved in 15 mL of 10 mMKH₂PO₄ and was filter sterilized. Five milligrams of sterile fungalelicitor dissolved in 333 μL 10 mM KH₂PO₄ was added per flask. Cellswere harvested 15 h after addition of elicitor. The same suspensionculture conditions were used before and after elicitor treatment. Cellswere recovered using a Nalgene PES filter unit (0.2 μm) followed by 3minutes of air flow. Filtered cells were immediately frozen in liquidnitrogen and kept at −76° C. until used. Non-elicitor-treated cells werehandled in the same manner, except for the addition of elicitor.

Microsome Preparation from Soybean Hypocotyls and Suspension-CulturedCells

For preparation of the crude extracts, 3 to 5 g of previously frozen,elicitor-treated and non-treated soybean hypocotyls and elicitor-treatedand non-treated suspension cultured cells were ground in liquid nitrogenusing a pre-chilled pestle and mortar. The powder was added to 25 mL ofextraction buffer (buffer A: 0.1M Tris-HCl, pH 7.5, 14 mMβ-mercaptoethanol, 20% (w/v) sucrose and 0.8 g of Dowex 1X2 resin (mesh200-400)), and the slurry was stirred for 20 to 30 minutes in anice-water bath. The slurry was transferred to Nalgene Oak Ridge tubesand centrifuged at 8000 g for 10 minutes at 4° C. The supernate wascarefully transferred into 13 mL polyallomer tubes which fit into aSorvall TH641 rotor and centrifuged at 160,000 g for 40 minutes to 2 hat 4° C. The precipitated microsomes were washed twice with the storagebuffer (buffer B: 80 mM KH₂PO₄, pH 8.5, 14 mM β-mercaptoethanol, 30%(v/v) glycerol) and resuspended with storage buffer. The microsomalpellet was gently homogenized by hand using a disposable plastic pestle,and the suspension was divided into several aliquots which were frozenon dry-ice. Bradford protein micro assays were used to quantify theprotein content of the microsomal preparations (Bio-Rad, Richmond,Calif.). Two microliters of a microsome preparation were diluted with198 μL of distilled water. Forty microliters of this dilution was mixedwith 10 μL of Bio-Rad protein assay solution in a microtiter plate, andthe total protein concentration was determined by reading the sample ina kinetic microplate reader (Molecular Devices Inc.), according to themanufacturer's instructions (Bio-Rad). Microsomes were stored at −76° C.until used.

Example 2 Development of Isoflavone Synthase Assay

An assay to measure isoflavone synthase activity was developed usingeither of the two substrates of isoflavone synthase, (±) naringenin(4′,5,7-trihydroxyflavanone; Sigma, N-5893) or liquiritigeninmonohydrate (4′,7-dihydroxyflavanone; Indofine, 02-1150S), dissolved in80% ethanol. The reaction mixture was prepared at room temperature andconsisted of 100 μM naringenin or liquiritigenin, 80 mM K₂HPO₄, 0.5 mMglutathione (Sigma, G-4251), 20% w/v sucrose, and 30 to 150 μg ofmicrosome preparation. The reaction mixtures were preincubated for 5minutes without NADPH (synthesis of genistein and daidzein requiresNADPH as a co-factor). The volume of microsomes and substrate added toany one reaction did not exceed 5% and 1%, respectively, of the totalreaction volume. A typical reaction volume was 250 μL. The reaction wasstarted by the addition of 40 nmol of NADPH per each 100 μL of finalreaction volume. The pH of the reaction mixture was 8.0 before theaddition of the substrate, NADPH and microsomes.

Microsomes were thawed, an aliquot removed and the remaining sample wasimmediately frozen on dry ice and stored in the freezer. The reactionsusing microsomes prepared from soybean elicitor-treated hypocotyls wererun for incubation periods of up to 24 h, while the reactions using theyeast microsomes were allowed to run for incubation periods of up to 14h. Following incubation, 200 μL of ethyl acetate was added directly tothe mixture and the mixture was shaken for 1 minute using a vortexmixer. Separation of the organic phase was accelerated by centrifugationfor 2 minutes at 4° C. The organic phase was removed and analyzed.

Qualitative and quantitative analyses were performed using a HewlettPackard 1100 series HPLC and a Hewlett-Packard/Micromass LC/MS. Sampleswere assayed on a Hewlett Packard 1100 series HPLC system using either aLi-Chrospher 100 RP-18 column (5 μm) or a Phenomenex Luna 3u C18 (2)column (150×4.6 mm). Using either column, samples from in vitromicrosome assays in ethyl acetate, were isocratically separated for 5minutes employing 65% methanol as the mobile phase. The second columnwas used for plant samples where the ethyl acetate was evaporated andthe samples resuspended in 80% methanol. In these cases separation useda 10 minutes linear gradient from 20% methanol/80% 10 mM ammoniumacetate, pH 8.3 to 100% methanol using a flow rate of 0.8 ml per minute.Genistein and daidzein were monitored by the absorbance at 260 mm andnaringenin and liquiritigenin were monitored by the absorbance at 280nm. Peak areas were converted to nanograms using, as standards forcalibration, authentic naringenin, liquiritigenin, genistein, anddaidzein (Indofine Chemical Company, Inc., Somerville, N.J.) dissolvedin ethanol.

Analyses using LC/MS employed 10 μL of the ethyl acetate phase that hadbeen first evaporated with nitrogen gas and resuspended in 100 μL of 25%acetonitrile in water. These samples were analyzed by aHewlett-Packard/Micromass LC/MS instrument. A twenty-five microlitersample was run on a Zorbax Eclipse XDB-C8 reverse-phase column (3×150mm, 3.5 micron) isocratically with 25% of solvent B in solvent A.Solvent A was 0.1% formic acid in water, and solvent B was 0.1% formicacid in acetonitrile. Mass spectrometry was carried out by electro-sprayscanning from 200-400 m/e, using +60 volt cone voltage. The diode arraysignals were monitored between 200-400 nm in both instruments.

The genistein and liquiritigenin signals observed in the in vitro assaysamples were verified by comparisons of retention time, diode arraydetected absorption spectra and mass spectrometry data to the standards.FIG. 2 presents the results of HPLC analyses of naringenin standards andFIG. 3 presents the results of HPLC analyses of genistein standards.

Incubations in the absence of an essential component required forisoflavone synthase-catalyzed synthesis of isoflavonoid (e.g., NADPH,naringenin, liquiritigenin, or microsomes) were performed as negativecontrols.

Positive control samples consisting of soybean microsomes which wereprepared from elicitor-treated hypocotyls and suspension culture cellswere used to establish the in vitro assay system. Optimization of thisin vitro assay system was critical for validation of the yeastexpression system for functional cloning. We observed positive results(i.e., the synthesis of genistein) in assays that used either themicrosomes of elicitor-treated soybean hypocotyls (FIG. 4) or thoseobtained from elicitor-treated cell suspension cultures (FIG. 6). Weobserved about six times higher specific enzyme activities of isoflavonesynthase in the microsomes of elicitor-treated hypocotyls and cellcultures (FIG. 4 and FIG. 6, respectively) than in the microsomesobtained from non-treated hypocotyls and cell cultures (FIG. 5 and FIG.7, respectively).

Example 3 Composition of Soybean cDNA Library, Isolation and Sequencingof cDNA Clone

A cDNA library was prepared using mRNAs from soybean seeds that had beenallowed to germinate for 4 hours. The library was prepared in Uni-ZAP™XR vector according to the manufacturer's protocol (Stratagene CloningSystems, La Jolla, Calif.). Conversion of the Uni-ZAP™ XR library into aplasmid library was accomplished according to the protocol provided byStratagene. Upon conversion, cDNA inserts were contained in the plasmidvector pBluescript. cDNA inserts from randomly picked bacterial coloniescontaining recombinant pBluescript plasmids were amplified viapolymerase chain reaction using primers specific for vector sequencesflanking the inserted cDNA sequences or plasmid DNA was prepared fromcultured bacterial cells. Amplified insert DNAs or plasmid DNAs weresequenced in dye-primer sequencing reactions to generate partial cDNAsequences (expressed sequence tags or “ESTs”; see Adams, M. D. et al.(1991) Science 252:1651-1656). The resulting ESTs were analyzed using aPerkin Elmer Model 377 fluorescent sequencer.

Example 4 Identification and Characterization of a cDNA Clone forIsoflavone Synthase

ESTs encoding candidate isoflavone synthases were identified byconducting BLAST (Basic Local Alignment Search Tool; Altschul, S. F., etal., (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/) searches for similarity to sequencescontained in the BLAST “nr” database (comprising all non-redundantGenBank CDS translations, sequences derived from the 3-dimensionalstructure Brookhaven Protein Data Bank, the last major release of theSWISS-PROT protein sequence database, EMBL, and DDBJ databases). ThecDNA sequences obtained in Example 3 were analyzed for similarity to allpublicly available DNA sequences contained in the “nr” database usingthe BLASTN algorithm provided by the National Center for BiotechnologyInformation (NCBI). The DNA sequences were translated in all readingframes and compared for similarity to all publicly available proteinsequences contained in the “nr” database using the BLASTX algorithm(Gish, W. and States, D. J. (1993) Nature Genetics 3:266-272) providedby the NCBI.

The insert in cDNA clone sgs1c.pk006.o20 was identified as a candidateisoflavone synthase gene by a BLAST search against the NCBI database.The 5′ sequence of this insert was determined to be related to Glycinemax cytochrome P450 monooxygenase CYP93C1p (CYP93C1) mRNA, the completecoding sequence of which may be found as NCBI General Identifier No.2739005. The CYP93C1p cDNA sequence was obtained using random isolationand screening to identify soybean P450s involved in herbicide metabolism(Siminszky B., et al. (1999) Proc. Natl. Acad. Sci. USA. 96:1750-1755).Isoflavone synthase catalyzes in soybeans the oxidation of7,4′dihyroxyflavanone (liquiritigenein) or 5,7,4′trihydroxyflananone(naringenin) to daidzein or genistein respectively. Earlier publishedwork (Kochs and Griesbach (1986) Eur. J. Biochem 155:311-318; Hashim etal. (1990) FEBS 271:219-222) suggested that the enzyme that catalyzesthis reaction is a cytochrome P450. Accordingly, in order to confirm theidentity of the polypeptide encoded by the insert in cDNA clonesgs1c.pk006.o20 as an isoflavone synthase, the polypeptide encoded bythis insert was evaluated for its ability to catalyze the formation ofgenistein from naringenin.

The ability of the cDNA insert in clone sgs1c.pk006.o20 to encode anisoflavone synthase was evaluated by expression of the encodedpolypeptide in an engineered yeast (Saccharomyces cerivisae) strain.Microsomes prepared from the engineered yeast strain transformed with aplasmid encoding the putative isoflavone synthase were assayed for theirability to mediate the synthesis of genistein in the presence ofsubstrate (naringenin).

Yeast strain W303-1B was used as the starting material and modified byhomologous recombination. The coding sequence of the P450 reductase HT1isolated from Helianthus tuberosus (NCBI General Identifier No. 1359894)was inserted into the integrative plasmid pYeDP110 (Pompon, D. et al.(1996) Meth. Enz. 272:51-64). Insertion was achieved after PCRamplification for addition of Bam HI and Eco RI restriction sites 5′ and3′ of the coding region, respectively, using the primers listed as SEQID NO:3 and SEQ ID NO:4. [SEQ ID NO:3] 5′-CGGGATCCATGCAACCGGAAACGGTCG-3′[SEQ ID NO:4] 5′-CCGGAATTCTCACCAAACATCACGGAGGTATG-3′

Transformation of W303-1B with the linearized plasmid led to homologousrecombination with the promoter and terminator sequences of theendogenous yeast reductase (CPR1) resulting in the disruption of theCPR1 gene and replacement with the URA3 gene and HT1 under the controlof the galactose-inducible promoter GAL10-CYC1. The resulting strain isdesignated WHT1.

Plasmid DNA (200 ng) from cDNA clone sgs1c.pk006.o20 was used astemplate for PCR with primers that are homologous to the vectorsequences flanking the cDNA cloning site (SEQ ID NO:5 and SEQ ID NO:6).[SEQ ID NO:5] 5′-TCAAGGAGAAAAAACCCCGGATCCATGTTGCTGGAACTTGCAC TTGG-3′[SEQ ID NO:6] 5′-GGCCAGTGAATTGTAATACGACTCACTATAGGGCG-3′

Amplification was performed using the GC melt kit (Clontech) with a 1 Mfinal concentration of GC melt reagent. Amplification took place in aPerkin Elmer 9700 thermocycler for 30 cycles as follows: 94° C. for 30seconds, 60° C. for 30 seconds, and 72° C. for 1 minute. The amplifiedinsert was then incubated with a modified pRS315 plasmid (NCBI GeneralIdentifier No. 984798; Sikorski, R. S. and Hieter, P. (1989) Genetics122:19-27) that had been digested with Not I and Spe I. Plasmid pRS315had been previously modified by the insertion of a bidirectional gal1/10 promoter between the Xho I and Hind III sites. The plasmid was thentransformed into the WHT1 yeast strain using standard procedures. Theinsert recombines though gap repair to form the desired plasmid (Hua, S.B., et al. (1997) Plasmid 38:91-96.). The resulting transformed yeaststrain is named Isoflavone Synthase GM1 (hereinafter referred to as“GM1”), and bears ATCC Accession No. 203606.

Yeast microsomes were prepared according to the methods of Pompon et al.(Pompon, D., et al. (1996) Meth. Enz. 272:51-64). Briefly, a yeastcolony was grown overnight (to saturation) in SG (-Leucine) medium at30° C. with good aeration. A 1:50 dilution of this culture was made into500 mL of YPGE medium with adenine supplementation and allowed to growat 30° C. with good aeration to an OD₆₀₀ of 1.6 (24-30 h). Fifty mL of20% galactose was added, and the culture was allowed to grow overnightat 30° C. The cells were recovered by centrifugation at 5,500 rpm forfive minutes in a Sorvall GS-3 rotor. The cell pellet was resuspended in80 mL of TEK buffer (0.1M KCl in TE) and left at room temperature forfive minutes. The cells were recovered by centrifugation as describedabove. The cell pellet was resuspended in 5 mL of TES-B (0.6M sorbitolin TE), and glass beads (0.5 mm diameter) were gently added until theyreached the surface of the suspension. The cells were disrupted byshaking up and down for five minutes, with an agitation frequency of atleast once every 0.5 second. Five mL of TES-B were added to the crudeextract, and the beads were washed with some agitation. The supernatantwas withdrawn and saved. The wash was repeated twice and the liquidfractions were pooled. The combined fractions were clarified by spinningat 11,000 rpm in a Sorvall SS34 rotor. The pellet was discarded and themicrosomes were precipitated by the addition of NaCl to a finalconcentration of 0.15 M. PEG 4000 was added to a final concentration of0.1 g/mL. The mixture was incubated on ice for at least 15 minutes, andthe microsomal fraction was recovered by at 8,500 rpm for 10 minutes inan SS34 rotor. The pellets were resuspended in TEG (glycerol, 20% byvolume, in TE) at a concentration of 20-40 mgs of protein per mL atwhich point they may be stored at −70° C. for months without anydetectable loss of activity.

Example 5 Demonstration of Functional Expression of Isoflavone Synthasein Yeast

The synthesis of genistein or daidzein from either naringenin orliquiritigenin was observed in an in vitro assay that was mediated byyeast microsomes prepared from the yeast transformant GM1 expressing thepolypeptide encoded by the insert in soybean cDNA clone sgs1c.pk006.o20.Samples were prepared and run on a LiChrospher 100 RP-18 column (5 μm)or a Phenomenex Luna 3u C18 (2) column (150×4.6 mm) as described inExample 2. Peaks in the yeast microsome assay samples were identified asbeing genistein or daidzein by their HPLC retention time and absorptionspectrum. The retention time and the absorption spectrum of the peakfound in the expected location of genistein was identical to theretention time and spectrum of authentic genistein (compare FIGS. 3 and4, FIGS. 17 and 18). The daidzein peak also had identical retention timeand absorption spectrum to the standard. More direct evidence wasobtained using LC/MS. Data for daidzein is shown in FIG. 19. Themolecular weights of the materials corresponding to the expectedgenistein and daidzein peaks from the yeast microsome assay samples were270.32 and 255.2, respectively. The molecular weights of authenticgenistein and daidzein are 270.23 and 255.2, respectively.

The synthesis of genistein in yeast microsomes obtained from the yeaststrain Isoflavone Synthase GM1 was monitored over the course ofincubation with the substrate naringenin. Samples representingincubation periods of 0 minutes and 1, 2, 3, 4 and 14 h were analyzed.Results are presented in FIGS. 8 through 13. A simultaneous increase ofgenistein, the product, and decrease of naringenin, the substrate ofisoflavone synthase, was observed. A detectable amount of genistein wassynthesized as early as 40 minutes (FIG. 14). Incubation of microsomeswith either naringenin or liquiritigenin as substrate shows an increasein accumulation of genistein and daidzein (the product) over ten hoursas seen in FIG. 26.

Genistein synthesis corresponds quantitatively with the amount of inputGM1 microsomes (FIG. 14 and FIG. 15). The genistein peak in the assayusing GM1 as a source was about 10 times higher than the peak observedfrom soybean microsome prepared from elicitor-treated hypocotyls(compare FIG. 4 and FIG. 13). Genistein synthesis by yeast microsomesusing GM1 also demonstrated an absolute requirement for NADPH. Withoutthe cofactor, the reaction mixture did not synthesize any detectablegenistein over a 4-h incubation (FIG. 16).

An unidentified peak, designated “peak 2,” with a retention time of1.59, was also detected during monitoring of reactions catalyzed byyeast microsomes at 280 nm (see FIG. 9 to FIG. 15). This peak was notsignificant in negative controls (FIG. 8 and FIG. 16). Koch andGrisebach proposed a hypothesis for the synthesis of an intermediateduring the conversion of naringenin to genistein (Kochs, G. andGrisenbach, H. (1985) Eur. J. Biochem. 155:311-318). This proposalstated that the oxidative aryl migration required to convert naringeninto genistein proceeds via a cytochrome P450 monooxygenase-mediatedconversion of the 2S-flavanone to a 2-hydroxyisoflavone, followed bydehydration to the isoflavonoid, possibly mediated by a solubledehydratase. The 2-hydroxyisoflavone intermediate was described asunstable and could spontaneously convert to genistein. In electrosprayLC/MS the most prominent peak in the spectrum of “peak 2” is at m/z=289,consistent with it being the [MH]⁺ form of the proposed hydroxylatedintermediate. The height of “peak 2” detected in the 4 h incubationsample was bigger than that for “peak 2” in the 14 h incubation sample.That sample showed the largest genistein peak among the microsome assaysthat were performed. It is suspected that “peak 2” may represent thisproposed intermediate that may be formed transiently during thesynthesis of genistein by isoflavone synthase. A similar intermediate(at m/z=273) was also detected in the conversion of liquiritigenin todaidzein (FIG. 19).

To compare the rates of genistein and daidzein synthesis by microsomesof the yeast transformant GM1, samples representing incubation periodsof 2, 4, 6, 8 and 10 h were analyzed. The peak areas for genistein anddaidzein were quantitated by calibration with authentic genistein anddaidzein standards. Assays were repeated three times and the averageamount of isoflavonoid synthesized at each time point was plotted, withvertical lines representing error bars (FIG. 26).

Example 6 Identification of CYP93C1 as a Soybean Isoflavone Synthase

The sequence of the mRNA encoding CYP93C1, a cytochrome P450monooxygenase, is found in the NCBI database having General IdentifierNo. 2739005. The function of the protein encoded by this mRNA has yet tobe identified. The cDNA insert in clone sgs1c.pk006.o20 encodes anisoflavone synthase and has sequence similarities with CYP93C1. Todetermine whether CYP93C1 encodes a functional isoflavone synthase, cDNAwas prepared and cloned into the yeast vector pRS315-gal and transformedinto yeast strain WHT1 to assay for its ability to produce genistein.The CYP93C1 mRNA was amplified from RNA isolated from soybean tissue(cv. S1990) infected with the fungal pathogen Sclerotinia slerotiorumusing RT-PCR. Fungal infection causes an increase in the amount ofisoflavonoid produced and thus the amount of isoflavone synthasetranscript was increased in the infected tissue. Soybean plants wereinfected 45 days after planting seeds and were harvested two days later.Total RNA was prepared using the TRIzol Reagent following themanufacturer's instructions (Gibco BRL) and 1 μg of the resulting totalRNA was converted into a first strand cDNA using the Superscript™Preamplification system and using oligodT as the reverse transcriptionprimer. One microliter of first strand cDNA was amplified by PCR usingthe primers listed as SEQ ID NO:7 and SEQ ID NO:8:5′-AAAATTAGCCTCACAAAAGCAAAG-3′ [SEQ ID NO:7]5′-ATATAAGGATTGATAGTTTATAGTAGG-3′ [SEQ ID NO:8]

The nucleotide sequence in SEQ ID NO:7 corresponds to nucleotides 3 to26 of the sequence found in NCBI General Identifier No. 2739005. Thenucleotide sequence in SEQ ID NO:8 corresponds to the complement ofnucleotides 1798 to 1824 of the sequence found in NCBI GeneralIdentifier No. 2739005. Amplification was performed on a Perkin ElmerApplied Biosystems GeneAmp PCR System using the Advantage-GC cDNApolymerase mix (Clontech), following the manufacturer's instructions,with a 1 M final concentration of GC melt reagent. Previous toamplification, the mixture was incubated at 94° C. for 5 minutes.Amplification was performed using 30 cycles of: 94° C. for 30 seconds,53° C. for 30 seconds and 72° C. for 2 minutes. Following amplification,the mixture was incubated at 72° C. for 7 minutes. The amplified productwas then cloned into pCR2.1 using “The Original TA Cloning Kit”(Invitrogen). Plasmid DNA was purified using QIAFilter cartridges(Qiagen Inc) according to the manufacturer's instructions. Sequence wasgenerated on an ABI Automatic sequencer using dye terminator technologyand using a combination of vector and insert-specific primers. Sequenceediting was performed using DNAStar (DNASTAR, Inc.). The sequencegenerated represents coverage at least two times in each direction. Thesequence of the resulting clone, presented in SEQ ID NO:9, was identicalwith that of CYP93C1 (NCBI General Identifier No. 2739005); the deducedamino acid sequence of this cDNA is shown in SEQ ID NO:10.

The above plasmid was then cloned into the yeast vector pRS315-gal usinggap repair as described in Example 4. Standard procedures were used totransform the resulting plasmid into the WHT1 yeast strain. Microsomeswere prepared from the WHT1 yeast strain containing the soybean CYP93C1sequence and assayed for the production of genistein and daidzein asdescribed in Example 5. The resulting microsomes exhibited isoflavonesynthase activities. To compare the rates of genistein and daidzeinsynthesis by microsomes of the yeast transformant containing the soybeanCYP93C1 sequence, samples representing incubation periods of 2, 4, 6, 8and 10 h were analyzed. The peak areas for genistein and daidzein werequantitated by calibration with authentic genistein and daidzeinstandards as prepared in Example 2. Daidzein and genistein accumulatedlinearly over the time course.

Example 7 Amplification and Identification of Isoflavone Synthase FromOther Legume Species

Nucleic acid sequences encoding isoflavone synthases from lupine, mungbean, snow pea, alfalfa, red clover, white clover, hairy vetch andlentil were derived from total RNA prepared from young seedlings. Mungbean sprouts and snow pea sprouts were obtained from the local grocerystore. Seeds for alfalfa, red clover, white clover, hairy vetch, andlentil were obtained from Pinetree Garden Seeds while seeds for lupine(cv Russell Mix) were obtained from Botanical Interests, Inc. Seedlingswere germinated in a controlled temperature growth chamber (14 h lightat 25° C. and 10 h dark at 21° C.) and harvested after approximately twoweeks except for lupine, which was harvested after approximately threeweeks. Total RNA was prepared using TRizol Reagent (Gibco BRL) accordingto the manufacturer's instructions. For each plant, a first strand cDNAwas prepared from 1 μg total RNA using the Superscript™ PreamplificationSystem (Gibco BRL) following the manufacturer's instructions. OligodTwas used as the reverse transcription primer in all cases except whiteclover where random hexamers were used.

Amplification was performed on a Perkin-Elmer Applied Biosystems GeneAmpPCR System 9700PCR using Advantage-GC cDNA polymerase mix (Clontech)according to the manufacturer's instructions and with a finalconcentration of GC melt reagent equal to 1 M. Amplification waspreceded in all cases by incubation at 94° C. for 5 minutes and wasfollowed by incubation at 72° C. for 7 minutes. Two sets of primers wereused for PCR amplification. Primer set one is composed of SEQ ID NO:11and SEQ ID NO:12 and primer set two is composed of SEQ ID NO:13 and SEQID NO:14: 5′-ATGTTGCTGGAACTTGCACTT-3′ [SEQ ID NO:11]5′-TTAAGAAAGGAGTTTAGATGCAACG-3′ [SEQ ID NO:12]5′-TGTTTCTGCACTTGCGTCCCAC-3′ [SEQ ID NO:13] 5′-CCGATCCTTGCAAGTGGAACAC-3′[SEQ ID NO:14]

The initial amplification of all samples was done using 1 μL of firststrand cDNA and primer set one (SEQ ID NO:11 and SEQ ID NO:12).Amplification of mung bean was performed using 30 cycles of 94° C. for30 seconds, 48° C. for 30 seconds and 72° C. for 2 minutes.Amplification of red clover was performed using 30 cycles of 94° C. for30 seconds, 50° C. for 30 seconds and 72° C. for 1 minute. Amplificationof white clover, lentil, hairy vetch, alfalfa and lupine was carried outin two steps. The first amplification reaction was performed using 30cycles of 94° C. for 30 seconds, 50° C. for 30 seconds and 72° C. forone minute. A second amplification reaction was done with 1 μL of theresulting product and primer set two (SEQ ID NO:13 and SEQ ID NO:14)using 30 cycles of 94° C. for 30 seconds, 50.5° C. for 30 seconds and72° C. for one minute. Amplification of snow pea was performed in threedifferent PCR reactions. The first reaction was performed using 30cycles of 94° C. 30 seconds, 50.5° C. for 30 seconds and 72° C. for oneminute. One microliter from the resulting product was used for a secondamplification reaction using primer set one and 30 cycles of 94° C. for30 seconds, 60° C. for 30 seconds and 72° C. for one minute. Theresulting reaction was analyzed on a 1% agarose gel and the band at theexpected size was gel purified using the QIAquick Gel Extraction Kit(Qiagen). The purified DNA was resuspended in 30 μL of water and 1 μLwas used as a template for a third PCR reaction using primer set onewith 30 cycles of 94° C. for 30 seconds, 60° C. for 30 seconds and 72°C. for 90 seconds.

The resulting mung bean, red clover and snow pea PCR sequences werecloned into pCR2.1 using “The Original TA Cloning Kit” (Invitrogen). Theresulting white clover, lentil, hairy vetch, alfalfa and lupine PCRsequences were cloned into pCR2.1 using TOPO™ TA Cloning Kit(Invitrogen). Plasmid DNA was purified using QIAFilter cartridges(Qiagen Inc) or Wizard Plus Minipreps DNA Purification System (Promega)following the manufacturer's instructions. Sequence was generated on anABI Automatic sequencer using dye terminator technology and using acombination of vector and insert-specific primers. Sequence editing wasperformed using DNAStar (DNASTAR, Inc.). All sequences representcoverage at least two times in both directions.

The nucleotide sequence of comprising the cDNA insert in clone alfalfa 1is shown in SEQ ID NO:15; the deduced amino acid sequence of this DNA isshown in SEQ ID NO:16. The nucleotide sequence comprising the cDNAinsert in clone alfalfa 2 is shown in SEQ ID NO:57; the deduced aminoacid sequence of this DNA is shown in SEQ ID NO:58. The nucleotidesequence comprising the cDNA insert in clone alfalfa 3 is shown in SEQID NO:59; the deduced amino acid sequence of this DNA is shown in SEQ IDNO:60. The nucleotide sequence comprising the cDNA insert in clone hairyvetch 1 is shown in SEQ ID NO:17; the deduced amino acid sequence ofthis DNA is shown in SEQ ID NO:18. The nucleotide sequence comprisingthe cDNA insert in clone lentil 1 is shown in SEQ ID NO:19; the deducedamino acid sequence of this DNA is shown in SEQ ID NO:20. The nucleotidesequence comprising the cDNA insert in clone lentil 2 is shown in SEQ IDNO:21; the deduced amino acid sequence of this DNA is shown in SEQ IDNO:22. The nucleotide sequence comprising the cDNA insert in clone mungbean 1 is shown in SEQ ID NO:23; the deduced amino acid sequence of thisDNA is shown in SEQ ID NO:24. The nucleotide sequence comprising thecDNA insert in clone mung bean 2 is shown in SEQ ID NO:25; the deducedamino acid sequence of this DNA is shown in SEQ ID NO:26. The nucleotidesequence comprising the cDNA insert in clone mung bean 3 is shown in SEQID NO:27; the deduced amino acid sequence of this DNA is shown in SEQ IDNO:28. The nucleotide sequence comprising the cDNA insert in clone mungbean 4 is shown in SEQ ID NO:29; the deduced amino acid sequence of thisDNA is shown in SEQ ID NO:30. The nucleotide sequence comprising thecDNA insert in clone red clover 1 is shown in SEQ ID NO:31; the deducedamino acid sequence of this DNA is shown in SEQ ID NO:32. The nucleotidesequence comprising the cDNA insert in clone red clover 2 is shown inSEQ ID NO:33; the deduced amino acid sequence of this DNA is shown inSEQ ID NO:34. The nucleotide sequence comprising the cDNA insert inclone snow pea 1 is shown in SEQ ID NO:35; the deduced amino acidsequence of this DNA is shown in SEQ ID NO:36. The nucleotide sequencecomprising the cDNA insert in clone white clover 1 is shown in SEQ IDNO:37; the deduced amino acid sequence of this DNA is shown in SEQ IDNO:38. The nucleotide sequence comprising the cDNA insert in clone whiteclover 2 is shown in SEQ ID NO:39; the deduced amino acid sequence ofthis DNA is shown in SEQ ID NO:40. The nucleotide sequence comprisingthe cDNA insert in clone lupine 1 is shown in SEQ ID NO:54; the deducedamino acid sequence of this DNA is shown in SEQ ID NO:55.

Plasmids corresponding to mung bean 2, red clover 2 and snow pea 1 wereamplified and the plant-specific DNA (corresponding to SEQ ID NO:25, SEQID NO:33 and SEQ ID NO:35) were transferred to the yeast vectorpRS315-gal following the gap repair method explained in Example 4 toproduce the yeast expression strains isoflavone synthase VR2, isoflavonesynthase TP2, and isoflavone synthase PS1, respectively. The eight aminoacids at the amino- and carboxy-terminus correspond to those translatedfrom the primers used in PCR amplification and not necessarily belong tothe endogenous genes. Microsomes were isolated from the resulting yeastWHT1 strains containing the mung bean, red clover or snow pea genes, andassayed for isoflavone synthase activity as described in Example 5, withminor modifications. After incubation for 16 hours, 200 μL of ethylacetate was added to recover the isoflavonoids from the assay solution,the ethyl acetate was evaporated under nitrogen using a heating moduleevaporation system and the sample resuspended in 200 μL of 80% methanol.A 10 μL sample of this solution was injected into a Phenomenex Luna 3μC18 (2) column (size: 150×4.6 mm. The samples were eluted over 10minutes using an increasing methanol gradient (from 20% methanol/80% 100mM ammonium acetate buffer (pH 5.9) to 100% methanol (v/v)) at a flowrate of 1 mL per minute. The levels of genistein and naringenin in theeluted samples were monitored through the absorption spectrum at 260 and290 nm. The genistein signal was verified by comparisons of retentiontime, diode array detected absorption spectra. As seen in Table 1,microsomes from all three strains produced genistein and thereforeexhibited isoflavone synthase activity. TABLE 1 Genistein SynthesisUsing in vitro Yeast Assay System Yeast expression strain GenisteinSynthesized Isoflavone Synthase VR2 1298 ng  Isoflavone Synthase TP2 59ng Isoflavone Synthase PS1 19 ng pRS315-gal Not detectable

Example 8 Amplification and Identification of Isoflavone Synthase FromNon-Legume Species

Isoflavonoids are most often found in the legumes, although there areoccasional examples of isoflavonoids in non-legume plants (Dewick, P.M., Isoflavonoids in The Flavonoids: Advances in Research edited by J.B. Harborne and T. J. Mabry pp. 535-640). To obtain isoflavone synthaseswith greater molecular diversity, isoflavone synthase genes from Betavulgaris (sugarbeet) were cloned and their activity tested. Sugarbeet, amember of the family Chenopodiaceae, is one of the few non-legumespecies to have been shown to have isoflavonoids present (Geigert, etal. (1973) Tetrahedron. 29:2703-2706).

Sugarbeet seeds were germinated in a growth chamber as described inExample 7 (14 h light at 25° C. and 10 h dark at 21° C.) and harvestedafter two weeks. Total RNA was prepared using TRIzol Reagent (Gibco BRL)according to the manufacturer's instructions. First strand cDNA wasprepared from 1 μg total RNA using the Superscript™ PreamplificationSystem (Gibco BRL) following the manufacturer's instructions withOligodT as the reverse transcription primer.

Amplification was performed on a Perkin-Elmer Applied Biosystems GeneAmpPCR System 9700PCR using Advantage-GC cDNA polymerase mix (Clontech)according to the manufacturer's instructions and with a finalconcentration of GC melt reagent equal to 1 M. Amplification waspreceded in all cases by incubation at 94° C. for 5 minutes and wasfollowed by incubation at 72° C. for 7 minutes.

Amplification was carried out in two steps. The first amplificationreaction was performed using 1 μL of first strand cDNA and primer setone (SEQ ID NO:11 and SEQ ID NO:12) with 30 cycles of 94° C. for 30seconds, 50° C. for 30 seconds and 72° C. for one minute. A secondamplification reaction was done with 1 μL of the resulting product withprimer set two (SEQ ID NO:13 and SEQ ID NO:14) and using 30 cycles of94° C. for 30 seconds, 50.5° C. for 30 seconds and 72° C. for oneminute. The resulting PCR sequence was cloned into pCR2.1 using TOPO™ TACloning Kit (Invitrogen). Plasmid DNA was purified using QIAFiltercartridges (Qiagen Inc) or Wizard Plus Minipreps DNA Purification System(Promega) following the manufacturer's instructions. Sequence wasgenerated on an ABI Automatic sequencer using dye terminator technologyand using a combination of vector and insert-specific primers. Sequenceediting was performed using DNAStar (DNASTAR, Inc.). All sequencesrepresent coverage at least two times in both directions. The nucleotidesequence comprising the cDNA insert in clone sugarbeet 1 is shown in SEQID NO:47; the deduced amino acid sequence of this DNA is shown in SEQ IDNO:48. The nucleotide sequence comprising the cDNA insert in clonesugarbeet 2 is shown in SEQ ID NO:61; the deduced amino acid sequence ofthis DNA is shown in SEQ ID NO:61.

The data in Table 2 summarizes the relationship of the isoflavonesynthase nucleotide and amino acid sequences disclosed herein. Reportedare the percent identity of the nucleotide sequences set forth in SEQ IDNOs:9, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 47 and 54 toinstant soybean isoflavone synthase sequence set forth in SEQ ID NO:1.In addition, the percent identity of the amino acid sequences deducedfrom the instant nucleotide sequences as set forth in SEQ ID NOs: 10,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 48 and 55 arecompared to the amino acid sequence set forth in SEQ ID NO:2. TABLE 2Percent Identity of Nucleotide Coding Sequences and Amino Acid Sequencesof Polypeptides Homologous to Isoflavone Synthase SEQ ID PercentIdentity NO. length to SEQ ID NO: 1/2 nt aa Crop (nts)* nucleotides (nt)amino acids (aa) 9 10 Soybean 1824 85.9 96.7 15 16 Alfalfa1 1501 99.599.0** 56 57 Alfalfa2 1501 92.2 96.2** 58 59 Alfalfa3 1501 92.3 96.6**17 18 Hairy vetch 1501 92.3 96.2** 19 20 Lentil1 1501 97.9 98.8** 21 22Lentil2 1501 92.3 96.4** 23 24 Mung bean1 1566 92.5 96.7 25 26 Mungbean2 1566 92.5 96.7 27 28 Mung bean3 1566 92.6 96.7 29 30 Mung bean41566 92.7 96.7 31 32 Red clover 1566 92.5 96.4 33 34 Red clover 156692.6 96.7 35 36 Snow pea 1563 99.3 99.0 37 38 White clover1 1496 99.398.4** 39 40 White clover2 1501 98.3 99.0** 60 61 Sugarbeet1 1497 91.995.6** 47 48 Sugarbeet2 1501 92.3 96.6** 54 55 Lupine 1501 92.2 96.2***SEQ ID NO: 1 contains 1756 nucleotides.**These sequences are 22 amino acids shorter because the primers usedfor PCR were derived from the soybean sequence.

The data presented in Table 2 indicates that the nucleotide and aminoacid sequences encoding the various isoflavone synthases are highlyconserved among divergent species. Sequence alignments and percentidentity calculations were performed using the Megalign program of theLASARGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).Multiple alignment of the sequences was performed using the Clustalmethod of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) withthe default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10).

A consensus sequence was determined by aligning the amino acid sequencesof the present invention using the Clustal method of alignment and thissequence is shown in SEQ ID NO:66. Amino acids not conserved areindicated by Xaa. These are: Xaa₁₀ Phe or Leu Xaa₁₆ Ser or Leu Xaa₂₃ Seror Thr Xaa₂₅ Ile or Lys Xaa₃₉ Lys or Arg Xaa₄₈ Pro or Leu Xaa₆₀ Pro orLeu Xaa₇₃ Leu or His Xaa₇₄ Ser or Tyr Xaa₉₅ Ala or Thr Xaa₉₆ Asn or HisXaa₁₀₂ Asn or Ser Xaa₁₁₀ Ile, Val, or Thr Xaa₁₁₂ Arg or His Xaa₁₁₇ Asnor Ser Xaa₁₁₈ Ser or Leu Xaa₁₂₁ Met or Arg Xaa₁₂₂ Ala or Val Xaa₁₂₄ Pheor Ile Xaa₁₂₉ Lys or Arg Xaa₁₄₇ Lys or Glu Xaa₁₅₉ Leu or Phe Xaa₁₆₂ Alaor Val Xaa₁₆₆ Ser or Gly Xaa₁₇₀ Gln or Arg Xaa₁₇₅ Val or Leu Xaa₁₈₃ Alaor Thr Xaa₁₈₇ Thr or Ile Xaa₁₉₁ Met or Val Xaa₂₀₉ Phe or Tyr Xaa₂₁₉ Argor Trp Xaa₂₂₃ Tyr or His Xaa₂₅₃ Gly or Glu Xaa₂₅₉ Lys or Glu Xaa₂₆₃ Valor Asp Xaa₂₆₄ Val, Asp, or Ile Xaa₂₆₈ Ala or Val Xaa₂₇₂ Phe or LeuXaa₂₈₅ Thr or Met Xaa₂₉₃ Glu or Asp Xaa₂₉₄ Thr, or Ile Xaa₃₀₁ Phe or LeuXaa₃₀₆ Thr or Ile Xaa₃₁₁ Val or Glu Xaa₃₁₂ Val or Ala Xaa₃₂₅ Arg or LysXaa₃₂₈ Gln or Glu Xaa₃₃₄ Val or Ala Xaa₃₄₂ Arg or Ile Xaa₃₇₇ Thr or IleXaa₃₈₁ Glu or Gly Xaa₃₈₅ Tyr, His, or Cys Xaa₃₈₇ Ile or Thr Xaa₃₉₃ Valor Ile Xaa₃₉₄ Leu or Pro Xaa₄₀₂ Arg or Lys Xaa₄₀₄ Ser or Pro Xaa₄₁₃ Seror Phe Xaa₄₂₂ Glu or Gly Xaa₄₂₈ Gly or Arg Xaa₄₂₉ Pro or Leu Xaa₄₃₅ Glnor Arg Xaa₄₄₇ Arg or Gly Xaa₄₅₃ Asn, Ser, or Ile Xaa₄₅₉ Met or Thr, andXaa₄₈₅ Asp or Gly

To verify that the similarity between the isoflavone synthase nucleotidesequences from soybean and from sugarbeet were not due to artifacts ofPCR, a nucleic acid sequence containing the soybean isoflavone synthaseset forth in SEQ ID NO:1 was used as a probe for Southern blot analysisagainst sugarbeet genomic DNA. Hybridization was done overnight at 65°C. in 6×SSC, 5× Denhardts. Filters were washed 2 times in 2×SSC, 1% SDSat room temperature and 2 times in 0.2×SSC, 0.5% SDS at 65° C.Hybridizing bands were detected indicating that sugarbeet does containgenes with high homology to the soybean isoflavone synthase sequence.

Example 9 Preparation of Transgenic Tobacco with Chimeric IsoflavoneSynthase Gene

The ability to obtain isoflavone synthase activity by expressing thegene from soybean clone sgs1c.pk006.o20 in other plants was tested bypreparing transgenic tobacco plants expressing the isoflavone synthasegene and assaying for genistein production. The 1.6 Kb isoflavonesynthase coding region from clone sgs1c.pk006.o20 (SEQ ID NO:1) wasamplified using a standard PCR reaction in a GeneAmp PCR System with theprimers shown in SEQ ID NO:41 and SEQ ID NO:42: [SEQ ID NO:41]5′-TTGCTGGAACTTGCACTTGGT-3′ [SEQ ID NO:42]5′-GTATATGATGGGTACCTTAATTAAGAAAGGAG-3′

The resulting DNA sequence (IFS) contains from the second codon to thestop codon of the soybean isoflavone synthase gene sequence followed bya Kpn I site. The following three sequences (in 5′ to 3′ order) wereassembled in pUC18 vector (New England Biolabs) to yield plasmid pOY160(depicted in FIG. 20):

-   -   35S/cabL, a promoter sequence comprising 1.3 Kb from the        cauliflower mosaic virus (CaMV) 35S promoter extending to 8 bp        downstream from the transcription start site followed by a 60 bp        leader sequence derived from the chlorophyll a/b binding protein        gene 22L (Harpster M. H. et al. (1988) Mol. Gen. Genet.        212:182-190);    -   IFS, the isoflavone synthase gene fragment generated by PCR        amplification using the primers from SEQ ID NO:41 and SEQ ID        NO:42.    -   Nos3′; an 800 bp fragment which contains the polyadenylation        signal sequence from the nopaline synthase gene (Depicker A. et        al. (19820 J. Mol. Appl. Genet. 1:561-573).

The 5′ end of IFS was ligated to Nco I-digested, filled-in, 35S/cabL.The 3′ end of IFS was digested with Kpn I and ligated to Kpn I-digestedNos3′.

The following three fragments were ligated to create plasmid pOY204:

-   -   1) The Hind III/Pst I fragment comprising the 35S/cabL-5′IFS        from pOY160,    -   2) The Pst I/Sal I fragment comprising the 3′IFS-Nos3′ from        pOY160,    -   3) The Hind III/Sal I fragment from vector pPZP211.

The vector pPZP211 contains an npt II gene fragment under the control ofthe 35S CaMV promoter conferring kanamycin resistance as the plantselectable marker (Hajdukiewicz P. et al. (1994) Plant Mol. Biol.25:989-994).

The plasmid pOY204 was transformed into the Agrobacterium tumefaciensstrain LBA4404 and was subsequently introduced into Nicotiana tobaccumby leaf disc co-cultivation following standard procedures (De Blaere etal. 1987 Meth. Enzymol. 143:277). The leaf discs were incubated forthree weeks on selection medium (MS salts with vitamins (Gibco BRL), 1mg/L 6-benzylaminopurine (BA), 100 mg/L kanamycin, and 500 mg/LClaforan). The regenerating plants were transferred to rooting medium(selection medium without BA) for another two weeks. Transformed plantswere identified by the appearance of roots in this selection media.Following standard protocols, DNA samples were prepared from sixrandomly-selected shoots and used as templates for PCR using the primersfrom SEQ ID NO:41 and SEQ ID NO:42. Verification of the presence of theisoflavone synthase coding region in the genome of the tested tobaccoshoots was done by separating the reaction product using a 1% agarosegel and staining with ethidium bromide. The expected 1.6 Kb fragment wasobtained as the reaction product in all the transgenic tobacco shootsand not in the untransformed tobacco controls.

Transcription of Soybean Isoflavone Synthase in Transgenic TobaccoShoots

Transcription of the isoflavone synthase gene in the transgenic tobaccoshoots was confirmed using RT-PCR. Total steady-state plant RNA wasextracted from four randomly-selected tobacco shoots resulting fromtransformation with pOY204 using the RNeasy Plant Mini Kit (Qiagen)following standard protocols. RT-PCR amplification was performed using“The SuperScript One Step RT-PCR Kit” (Gibco BRL) with the primers:5′-GACGCCTCACTTACGACAACTCTGTG-3′ [SEQ ID NO:43]5′-CCTCTCGGGACGGAATTCTGATGGT-3′ [SEQ ID NO:44]

After incubation at 50° C. for 45 minutes, amplification was carried outusing 37 cycles of 93° C. for 30 seconds, 64° C. for 30 seconds and 72°C. for 1 minute. The resulting DNA was separated on a 1% agarose gel.Samples from the putative isoflavone synthase-containing tobacco showedan 840 bp band not seen in the sample from the untransformed tobaccocontrol.

Example 10 Expression of Soybean Isoflavone Synthase in TransgenicTobacco

Activity of Soybean Isoflavone Synthase in Tobacco Shoots

The activity of the soybean isoflavone synthase in the transgenictobacco was determined by analyzing shoots for the presence ofgenistein. Approximately one gram of tissue from shoots of five-week-oldrooting transformants and from untransformed tobacco plants were groundin liquid nitrogen and extracted for 20 minutes at room temperatureusing 10 mL of 80% ethanol. After filtration through Acrodisc CR-PTFEsyringe filters (Gelman Sciences), 3 mL from each extraction solutionwere concentrated to 1 mL by evaporation under nitrogen gas flow using a50° C. heating block. To hydrolyze any malonyl or glucosyl-derivatizedcompounds present, 3 mL of 1 N HCl were added and the samples incubatedat 95° C. for 2 h followed by extraction using 1 mL ethyl acetate. Fivehundred μL of the ethyl acetate phase were dried under nitrogen andresuspended in 20 μL chloroform. The presence of genistein in thesamples was determined by gas chromatography/mass spectroscopy (GC/MS)analysis.

Before injection into a Hewlett Packard 6890 gas chromatograph, thehydroxyl groups in the samples were derivatized to trimethylsilylate bythe addition of 100 μL of BSTFA (N,O-bis(trimethylsilyl)-trifluoroacetamide; Supelco) and incubation at 37°C. for 1 h. The samples were dried under nitrogen gas and re-dissolvedin 20 μL chloroform immediately before manual injection into the gaschromatograph. Two μL of sample were manually injected onto a 15 meterdry bed GC capillary column (J&W, Jones Chromatography, Mid Glamorgan,UK) through an injector port operated in the split mode (5:1). Theinitial oven temperature was set at 200° C. and the column was set at alinear temperature gradient from 200° C. to 300° C. in 20 minutes with ahelium gas flow rate of 1.5 mL/minute. The mass spectrum was monitoredusing a Hewlett Packard 5973 mass-selective detector at an ionizationpotential of 70 eV. The mass ions identified from the cracking patternof pure genistein treated as mentioned above are 414 and 399 m/z. Thesepeaks represent the products of partially derivatized genistein, theform obtained following the above procedure. Twenty nine of thirty threetobacco transformants analyzed by gas chromatography had an identifiablegenistein peak at 8.7 minutes. The presence of genistein in these peakswas confirmed by the detection of peaks at 414 and 399 m/z in the massspectra. These results confirmed that the soybean isoflavone synthasecoding region is expressed in tobacco plants under control of the 35SCaMV promoter and causes novel production of genistein in tobacco shoottissue.

Presence of Genistein in Tobacco Flowers

Flowers from the tobacco transformants were assayed for the presence ofgenistein. Extracts were prepared as described above, except that afterhydrolysis, the dried ethyl acetate extracts were resuspended in 1 mL of80% methanol. The HPLC protocol was the same as in Example 2 using aPhenomenex Luna 3u C18 (2) column (150×4.6 mm). As compared to extractsfrom wild type plants, the transformant flowers contained two additionallarge peaks in the HPLC profile. One of these peaks was identified asgenistein while the other is unknown. Detection of the large genisteinpeak in the HPLC profile of the tobacco flower extracts indicated thatthere was a much higher amount of genistein present in the tobaccoflowers than in the tobacco shoots, since the genistein in the shootsamples was only detectable by GC/MS. The prevalence of genistein in theflowers relates to the expression of the anthocyanin biosyntheticpathway, which is active in the flowers as indicated by the pink flowercolor. An active anthocyanin pathway produces the naringenin substratefor isoflavone synthase.

Example 11 Expression of Soybean Isoflavone Synthase in TransgenicArabidopsis

Arabidopsis thaliana was transformed with the plasmid pOY204 via inplanta vacuum infiltration following standard protocols (Bechtold et al.(1993) CR Life Sciences 316:1194-1199). Briefly, three-week-oldArabidopsis thaliana ectotype WS plants were submerged in 500 mL ofAgrobacterium, strain GV3101 harboring pOY204, suspended in basic MSmedia (Gibco BRL) and vacuum was applied repeatedly for 10 minutes. Theinfiltrated plants were allowed to set seeds for another three weeks.The harvested seeds were surface-sterilized, then germinated and grownfor three weeks on plates containing 75 mg/L kanamycin. Approximately120 green healthy plants were recovered in the first round of screeningand were transferred to soil for two more weeks. The plants at thisstage had green immature pods and few leaves. Extracts were prepared andanalyzed by HPLC and GC/MS as described in Example 2, except that afterhydrolysis, the dried ethyl acetate extracts were resuspended in 1 mL of80% methanol. Five of twelve randomly-selected Arabidopsis transformantsanalyzed by HPLC had an identifiable genistein peak at 8.7 minutes. GCMS analysis confirmed the presence of genistein in these peaks bydetection of the characteristic peaks at 414 and 399 m/z in the massspectra. These results show that the soybean isoflavone synthase gene isfunctional in the Arabidopsis plants and genistein is produced.

Example 12 Enhancing Isoflavonoid Levels in Transgenic Arabidopsis

To determine whether activation of the phenylpropanoid pathway resultsin increased accumulation of isoflavonoids in IFS-transformedArabidopsis, the pathway was activated by UV light treatments.Homozygous Arabidopsis transformants of line A109-4, which synthesizegenistein, were identified through germination on kanamycin-containingmedium by first selecting a transformant that segregated kanamycinresistance in a 3:1 ratio. A resistant progeny from this generation thatthen produced 100% resistant progeny was identified as a homozygote.Plants from this population and wild type Arabidopsis plants weretransferred to 2-inch pots 10 days after germination and grown for 10more days. Plants were placed directly under 366 nm UV light for 16 h(46 mWatt/cm², using an UVL-56 BLAK-Ray Lamp from UV Products, Inc., SanGabriel, Calif.). Control plants were placed under the same describedenvironment except for the UV illumination. The above ground parts ofArabidopsis plants were pulverized in liquid nitrogen to fine powderimmediately after UV treatment. The tissues were extracted with 10 mL80% methanol per 1 gram of fresh weight. The genistein content fromtissue extracts of UV-treated and untreated plants was determined byHPLC using a Phenomenex Luna 3u (2) column (150×4.6 mm) and a mobilphase linear gradient which goes in 15 minutes from 20% methanol, 80% 10mM ammonium acetate, pH 8.3 to 100% methanol followed by 100% methanolfor 5 minutes as described in Example 2. Aliquots from the same extractswere also assayed for anthocyanin accumulation using photospectrometryas described by Bariola, P. A., et. al. ((1999) Plant Physiol.119:331-342). Briefly, one mL of extract was mixed with one mL of 0.5%(v/v) HCl followed by the addition of two mL of chloroform and vortexingfor ten seconds. The mixture was allowed to separate to two phases atroom temperature. The absorbance of the aqueous phase was assayed at 530nm and 657 nm. The anthocyanin content was calculated by subtracting theabsorbance value at 657 from the absorbance value at 530 and normalizingto fresh weight. As seen in Table 3, the anthocyanin content andgenistein level in IFS-transformed Arabidopsis varies with UV treatment(The average and standard deviations of four independent plants fromeach group are shown). TABLE 3 Anthocyanin Content and Genistein Levelsin Transgenic Arabidopsis Plants Anthocyanin Genistein (by HPLC)(A530-A657) (mAu/25 uL) Sample Control UV Control UV Control 0.0463 ±0.0148 0.0591 ± 0.0202 0 0 Plants (no IFS gene) A109-4 0.0339 ± 0.01000.0368 ± 0.0116 121 ± 41 303 ± 58 (35S-IFS)

Anthocyanins are products of one branch of the phenylpropanoid pathway,and the level of their accumulation is an indication of the activity ofthis pathway. As seen in the table above, genistein was not detectableand the anthocyanin levels increased by about 28% after UV treatment inthe control plants. In plants expressing IFS the anthocyanin levels werenot significantly increased while the genistein levels more thandoubled. A duplication of this experiment also showed an increase ingenistein level (anthocyanin levels without UV treatment:0.1426+/−0.0245; and with UV treatment: 0.1463+/−0.0145 (units asdescribed above); genistein without UV treatment: 602+/−94; and with UVtreatment: 857+/−46 (units as described above)). In this case the levelof anthocyanins in non-treated plants was much higher, probably due toinsect infestation. The level of genistein was higher in non-treatedplants and the increase with UV treatment was not as large as in thefirst experiment. These results demonstrate that activation of thephenylpropanoid pathway, in this case by stress treatment (UV or insectinfestation), results in an increased level of genistein accumulation intransformants expressing isoflavone synthase.

Example 13 Expression of Soybean Isoflavone Synthase in Monocot Cells

The ability to obtain isoflavone synthase activity in monocot cells wastested by transforming the soybean gene from clone sgs1c.pk006.o20 intocorn suspension cells and assaying for genistein production. The soybeanisoflavone synthase gene was cloned in a vector for expression inmonocot cells and its activity determined by the expression of genisteinin corn. A chimeric isoflavone synthase gene plasmid was prepared(pOY206) using the pGEM9Zf cloning vector (Promega) for expression ofthe instant isoflavone synthase in monocots. The following fragmentswere inserted between two copies of the 3 Kb SAR fragment (the Aelement, originally located between 8.7 and 11.7 kb upstream of thechicken lysozyme gene coding region (Loc P. V. and Stratling W. H.(1988) EMBO J. 7:655-664):

-   -   1. the 35S/cabL promoter fragment from Example 9,    -   2. a 490 bp fragment containing the sixth intron from the maize        Adh1 gene (Mascarenhas, D. et al. (1990) Plant Mol. Biol.        15:913-920) and ending with an Nco I site,    -   3. IFS, the isoflavone synthase fragment from Example 9,    -   4. a 285 bp fragment containing the polyadenylation signal        sequence from the nopaline synthase gene (Depicker A. et        al. (1982) J. Mol. Appl. Genet. 1:561-573).        Gene Combinations used for Corn Cell Transformation

The plasmid pOY206 (FIG. 21) containing the chimeric isoflavone synthasegene for expression in monocots was transformed into corn cells inconjunction with plasmid pDETRIC. Plasmid pDETRIC contains the bar genefrom Streptomyces hygroscopicus that confers resistance to the herbicideglufosinate (Thompson et al. (1987) EMBO J. 6:2519). In the pDETRICplasmid the bar gene is under the control of the CaMV 35S promoter, itstranslation-initiation codon has been changed from GTG to ATG for propertranslation initiation in plants (De Block et al. (1987) EMBO J.6:2513), and uses the Agrobacterium tumefaciens octopine synthasepolyadenylation signal.

Since the phenylpropanoid pathway is not active in corn suspension cellsa third plasmid containing a gene encoding a transcription factor thatactivates the phenylpropanoid pathway was, in some cases, bombarded intothe corn cells in conjunction with isoflavone synthase gene. Thisplasmid, pDP7951 (depicted in FIG. 22 and bearing ATCC accession numberPTA-371), contains in the 5′-3′ orientation:

-   -   the Agrobacterium nopaline synthase gene promoter region,    -   a tobacco mosaic virus (TMV) omega enhancer sequence,    -   the fifth intron from the maize adh1 gene,    -   CRC (a chimera containing the maize R region between the region        encoding the C1 DNA binding domain and the C1 activation        domain),    -   the potato protease inhibitor II polyadenylation signal        sequence.

Additionally, a chimeric gene consisting of the CRC coding regionexpressed from the CaMV 35S promoter was prepared and used in corn celltransformations. The Sma I fragment of DP7951 containing CRC was ligatedto Nco I and Kpn I ends that had been blunt ended with Mung beannuclease (New England Biolabs) to create the chimeric gene:35S/cabL-IFS-Nos3′. This plasmid is called pOY162, and its restrictionenzyme map is shown in FIG. 23.

Transformation of Monocot Cells

Black Mexican Sweet (BMS) suspension culture is a commonly used,corn-derived, monocot cell line. Cultures were maintained in MS2D medium(MS salts with vitamins (Gibco BRL), 20 g/L sucrose, 2 mg/L2,4-dichlorophenoxyacetic acid, pH 5.8), incubated with shaking (125rpm) at 26° C. in the dark, and subcultured with fresh medium every fivedays.

Transformations were performed by microprojectile bombardment using aDuPont Biolistic PDS 1000/He system (Klein T. M. et al. (1987) Nature327:70-73). Gold particles (0.6 microns) were coated with mixtures ofplasmid DNAs as indicated in Table 4: TABLE 4 Plasmid Groups used inMaize Transformations Group Plasmids 1 3 μg pDETRIC + 6 μg pOY206 2 3 μgpDETRIC + 6 μg pOY206 + 6 μg pDP7951 3 3 μg pDETRIC + 6 μg pDP7951 4 3μg pDETRIC + 6 μg pOY206 + 6 μg pOY162

Two days after subculture, BMS suspension culture aliquots (6 mL each),were evenly distributed over Whatman#1 filter disks, transferred ontosolid MS2D medium (MS2D, 7 g/L agar) and incubated at 26° C. overnight.Filter disks containing the BMS cells were positioned approximately 3.5inches away from the retaining screen and bombarded twice. Membranerupture pressure was set at 1,100 psi and the chamber was evacuated to−28 inches of mercury. Bombarded tissues were incubated for four days at26° C. in the dark and then transferred to MS2D selection medium (solidMS2D medium containing 3 mg/L Bialaphos). Resistant tissue wastransferred to fresh MS2D selection medium after seven weeks and tissuewas harvested for analysis two weeks later.

Analysis of Transformed Corn Cells for Synthesis of Anthocyanins andGenistein

All control tissue and BMS lines transformed with group 1 were white incolor. Approximately half of the Bialaphos-selected resistant tissuethat grew in plates bombarded with groups containing CRC (groups 2 and3) showed the wild type white color, while the other half showed variousdegrees of red coloration, a visual indication of anthocyaninaccumulation. The red phenotype indicates that expression of CRC inthese lines is sufficient to transcriptionally activate the expressionof genes in the phenylpropanoid pathway leading to anthocyanin synthesisand accumulation (Grotewold E. et al. (1998) Plant Cell 10:721-740).Presence of the isoflavone synthase gene in these tissues was confirmedby the appearance of the appropriate sized fragments when performing PCRon genomic DNA using primers from SEQ ID NO:43 and SEQ ID NO:44. Thepresence of the CRC coding region in these tissues was verified by theproduction of an appropriate fragment when performing PCR on genomic DNAusing the primers from SEQ ID NO:45 (to the R region) and SEQ ID NO:46(to the 3′ untranslated region from potato protease inhibitor II gene).5′-GCGGTGCACGGGCGGACTCTTCTTC-3′ [SEQ ID NO:45]5′-CGCCCAATACGCAAACCGCCTCTCC-3′ [SEQ ID NO:46]

Tissue from 25 lines transformed with Group 1, 5 white lines resultingfrom transformation with Group 2, 7 red lines transformed with Group 2,6 white lines transformed with Group 3, and 6 red lines transformed withGroup 3 was harvested and analyzed for the presence of genistein usingHPLC and GC-MS. Extracts were prepared and analyzed as described inExample 2. The genistein HPLC peak and the identifying 414 and 399 m/zMS peaks were detected in the extracts from all seven red linestransformed with Group 2 while no genistein was detected in any of thewhite lines transformed with the same plasmids. Lines transformed withGroup 3 did not have genistein whether they were red or white. Sixteenlines transformed with Group 4 also produced genistein. A summary ofthese results is shown in Table 5. TABLE 5 Genistein Synthesis inTransformed BMS Tissue Tissue Naringenin Genistein Group No. ColorProduced Produced 1 25 White NO NO 2 5 White NO NO 2 7 Red YES YES 3 6White NO NO 3 6 Red YES NO 4 16 Red YES YES

The synthesis of genistein in BMS lines transformed with a soybeanisoflavone synthase-containing construct indicated that the soybeanprotein was expressed and was functional in monocot cells. Genistein wasonly produced in cell lines producing naringenin indicating that thesoybean isoflavone synthase gene was only effective in the presence ofan activated phenylpropanoid pathway. The intermediate naringenin in thephenylpropanoid pathway provided the substrate for isoflavone synthaseto produce genistein.

Example 14 Synthesis of Daidzein in Monocot Cells

The activity of chalcone reductase determines the relative levels ofsubstrates available for isoflavone synthase to produce genistein ordaidzein (see FIG. 1). Chalcone reductase reduces4,2′,4′,6′-tetrahydroxychalcone to 4,2′,4′-trihydroxychalcone, thusproducing liquiritigenin as the substrate for isoflavone synthase toproduce daidzein. Chalcone reductases are present in legumes, but havenot been found in most non-legume plants including Arabidopsis, tobacco,and corn. To produce daidzein in non-legume plants, a plasmid DNAcontaining a soybean chalcone reductase gene was introduced into cornsuspension cells by microprojectile bombardment, together with aselection marker, CRC, and IFS constructs as described in Example 13.

A soybean cDNA clone encoding chalcone reductase was identified byhomology to known chalcone reductase genes of alfalfa (Ballance andDixon (1995) Plant Phys. 107:1027-1028). The cDNA library was preparedusing mRNAs from eight-day-old soybean roots inoculated with cystNematode for four days, and sequenced as described in Example 3. BLASTanalysis was performed as described in Example 4. The DNA containing theentire coding region from the identified clone, src3c.pk009.e4, wasamplified using PCR with the primers shown in SEQ ID NO:62 and SEQ IDNO:63 5′-GTTACCATGGCTGCTGCTATTG-3′ [SEQ ID NO:62]5′-TTAAACGTAAAATGAAACAAGAGG-3′ [SEQ ID NO:63]

The 5′ primer had an Nco I site at the start of the coding region. The1.3 kb PCR product was subcloned into the pTOPO2.1 vector (InvitrogenInc., Carlsbad, Calif.). The 1.3 kb coding region fragment was excisedas a Nco I/Kpn I fragment, using the Nco I site and the Kpn I site fromthe vector. This fragment was isolated and ligated between the 35S/CabLpromoter and Nos 3′polyadenylation signal sequence in the pUC18 vectoras described in Example 9, to produce plasmid pCHR40, which was used inthe BMS transformation experiments.

Transformation of corn suspension cells was done as described in Example13, using pDETRIC, pCHR40, pOY206 and pOY162. Selection and culturingwere as described in Example 13. Each selected line was assayed for thepresence of the IFS and CRC genes using PCR as in Example 13. Thepresence of the CHR gene was determined by the appearance of a 0.6 kbfragment when performing PCR on the tissues using the primers shown inSEQ ID NO:64 and SEQ ID NO:65: 5′-GACACTTCGACACTGCTGCTGCTTAT-3′ [SEQ IDNO:64] 5′-TCTCAAACTCACCTGGGCTATGGAT-3′ [SEQ ID NO:65]

Of 32 lines screened, five carried all three transgenes. Extracts wereprepared, as described in Example 13, from these 32 lines and a controlline that carries the CRC and IFS genes, but not the CHR gene. All ofthe extracts were treated with 1 N HCl to hydrolyze all possibleoligosaccharide derivatives as described in Example 10. HPLC and GC-MSwere performed as described in Examples 2 and 10. One out of the fivelines was shown to produce daidzein. In the HPLC assay, in addition tothe peaks of naringenin and genistein, a small peak occurred at the sameretention time as the daidzein standard (9.6 min) (FIGS. 27C and D).This peak was not present in the control samples (FIGS. 27A and B). Inthe GC-MS assay, the daidzein-specific cracking pattern was found at thesame retention time as the standard (8.0 min). All of the major ions ofthe daidzein spectrum were present (m/z: 398, 383, 218, 97). Thisexample shows that introduction of the soybean chalcone reductase geneinto corn cells together with the isoflavone synthase and CRC genesresults in the production of both daidzein and genistein.

Example 15 Alteration of Isoflavonoid Levels-in Soybean Somatic Embryos

The ability to change the levels of isoflavonoids by overexpressing thegene from soybean clone sgs1c.pk006.o20 in soybean somatic embryos wastested by preparing transgenic soybean somatic embryos and assaying theisoflavonoid levels. The entire insert from clone sgs1c.pk006.o20 (SEQID NO:1) was amplified in a standard PCR reaction on a Perkin ElmerApplied Biosystems GeneAmp PCR System using Pfu polymerase (Stratagene)with the primers shown in SEQ ID NO:49 and SEQ ID NO:50:5′-GAATTCGCGGCCGCTCTAGAACTAGTGGAT-3′ [SEQ ID NO:49]5′-GAATTCGCGGCCGCGAATTGGGTACCGGGC-3′ [SEQ ID NO:50]

The resulting fragment is bound by Not I sites in the primer sequencesand contains a 5′ leader sequence, the coding region for isoflavonesynthase, the untranslated 3′ region from SEQ ID NO:1, and a stretch of18 A residues at the 3′ end. This fragment was digested with Not I andligated to Not 1-digested and phosphatase-treated pKS67. The plasmidpKS67 was prepared by replacing in pRB20 (described in U.S. Pat. No.5,846,784) the 800 bp Nos 3′ fragment, described in Example 9, with the285 bp Nos 3′ fragment, described in Example 12. Clones were screenedfor the sense orientation of the isoflavone synthase insert fragment bydigestion with Bam HI. The resulting plasmid pKS93s, shown in FIG. 24,has the beta-conglycinin promoter operably linked to the fragmentencoding isoflavone synthase followed by the Nos 3′end. Plasmid pKS93scontains a T7 promoter/HPT/T7 terminator cassette for expression of theHPT enzyme in certain strains of E. coli, such as NovaBlue (DE3) (fromNovagen), that are lysogenic for lambda DE3 (which carries the T7 RNAPolymerase gene under lacV5 control). Plasmid pK93s also contains the35S/HPT/NOS 3′ cassette for constitutive expression of the HPT enzyme inplants. These two expression systems allow selection for growth in thepresence of hygromycin to be used as a means of identifying cells thatcontain plasmid DNA sequences in both bacterial and plant systems.

Transformation of Soybean Somatic Embryo Cultures

The following stock solutions and media were used for transformation andpropagation of soybean somatic embryos: Stock Solutions (g/L) MS Sulfate100x stock MgSO₄.7H₂O 37.0 MnSO₄.H₂O 1.69 ZnSO₄.7H₂O 0.86 CuSO₄.5H₂O0.0025 MS Halides 100x stock CaCl₂.2H₂O 44.0 KI 0.083 CoCl₂.6H₂O 0.00125KH₂PO₄ 17.0 H₃BO₃ 0.62 Na₂MoO₄.2H₂O 0.025 Na₂EDTA 3.724 FeSO₄.7H₂O 2.784B5 Vitamin stock myo-inositol 100.0 nicotinic acid 1.0 pyridoxine HCl1.0 thiamine 10.0 Media SB55 (per Liter) 10 mL of each MS stock 1 mL ofB5 Vitamin stock 0.8 g NH₄NO₃ 3.033 g KNO₃ 1 mL 2,4-D (10 mg/mL stock)0.667 g asparagine pH 5.7 SB103 (per Liter) 1 pk. Murashige & Skoog saltmixture* 60 g maltose 2 g gelrite pH 5.7 SB148 (per Liter) 1 pk.Murashige & Skoog salt mixture* 60 g maltose 1 mL B5 vitamin stock 7 gagarose pH 5.7*(Gibco BRL)

Soybean embryonic suspension cultures were maintained in 35 mL liquidmedia (SB55) on a rotary shaker (150 rpm) at 28° C. with a mix offluorescent and incandescent lights providing a 16 h day 8 h nightcycle. Cultures were subcultured every 2 to 3 weeks by inoculatingapproximately 35 mg of tissue into 35 mL of fresh liquid media.

Soybean embryonic suspension cultures were transformed with pKS93s bythe method of particle gun bombardment (see Klein et al. (1987) Nature327:70-73) using a DuPont Biolistic PDS 1000/He instrument. Five μL ofpKS93s plasmid DNA (1 g/L), 50 μL CaCl₂ (2.5 M), and 20 μL spermidine(0.1 M) were added to 50 μL of a 60 mg/mL 1 mm gold particle suspension.The particle preparation was agitated for 3 minutes, spun in a microfugefor 10 seconds and the supernate removed. The DNA-coated particles werethen washed once with 400 μL of 70% ethanol and resuspended in 40 mL ofanhydrous ethanol. The DNA/particle suspension was sonicated three timesfor 1 second each. Five μL of the DNA-coated gold particles were thenloaded on each macro carrier disk.

Approximately 300 to 400 mg of two-week-old suspension culture wasplaced in an empty 60 mm×15 mm petri dish and the residual liquidremoved from the tissue using a pipette. The tissue was placed about 3.5inches away from the retaining screen and bombarded twice. Membranerupture pressure was set at 1100 psi and the chamber was evacuated to−28 inches of Hg. Two plates were bombarded, and following bombardment,the tissue was divided in half, placed back into liquid media, andcultured as described above.

Fifteen days after bombardment, the liquid media was exchanged withfresh SB55 containing 50 mg/mL hygromycin. The selective media wasrefreshed weekly. Six weeks after bombardment, green, transformed tissuewas isolated and inoculated into flasks to generate new transformedembryonic suspension cultures.

Transformed embryonic clusters were removed from liquid culture mediaand placed on a solid agar media, SB103, containing 0.5% charcoal tobegin maturation. After 1 week, embryos were transferred to SB103 mediaminus charcoal. After 5 weeks on SB103 media, maturing embryos wereseparated and placed onto SB148 media. During maturation embryos werekept at 26° C. with a mix of fluorescent and incandescent lightsproviding a 16 h day 8 h night cycle. After 3 weeks on SB148 media,embryos were analyzed for the expression of the isoflavonoids. Eachembryonic cluster gave rise to 5 to 20 somatic embryos.

Non-transformed somatic embryos were cultured by the same method as usedfor the transformed somatic embryos.

Analysis of Transformed Somatic Embryos

At the end of the 8^(th) week on SB103 medium somatic embryos wereharvested from 12 independently transformed lines. Somatic embryos werecollected individually and stored in 96-well plates at −80° untillyophilized. Somatic embryos were lyophilized for 24 hours. Three tofive lyophilized somatic embryos were pooled in a micro centrifuge tubeand the dry weight was measured three times. Three samples of driedembryos were assayed for each transformed line. An 80% methanol solutionwas added to the lyophilized somatic embryos and the samples incubatedfor 24 h in the dark at room temperature to extract isoflavonoids. The80% methanol solution was filtered through a Costar nylon membranemicrocentrifuge filter with 0.22 μm pore size (Sigma).

For HPLC analysis of the extracts, twenty μl of the 80% methanol samplewas applied to a Phenomenex Luna 3μ C18 (2) column (size: 150×4.6 mm).Separation occurred during the gradient elution of 10 mM ammoniumbuffer, pH 8.35 (solvent A) and methanol (solvent B) as the mobilephase. Continuous increasing of solvent B in solvent A, from 20 to 100%for 10 min was employed. Standards for the isoflavonoids daidzin,daidzein, glycitin, glycitein, genistin, genistein, liquiritigenin andnaringenin were prepared by the gradual addition of 80% methanol to eachpowder. The peaks and spectra corresponding to daidzein, glycitin andgenistein conjugated with malonylated glucosides were determined byLC/MS. Isoflaovonoids were monitored through the absorption spectra at260 and 280 nm. The isoflavonoid signals observed in the soybean somaticembryo samples were verified by comparisons of the retention times anddiode array detected absorption spectra with those of the standards. Theareas of all peaks corresponding to the isoflaovones in a sample wereadded and divided by the dry weight of that sample. These dry weightbased normalized area sums were used for statistical analysis.

An analysis of variance test (ANOVA; Steel, R. G. D. and Torrie, J. H.(1996) Principles and Procedures of Statistics: A Biometrical Approach(McGraw-Hill Series in Probability and Statistics, New York) wasconducted using Microsoft Excel 97 (Microsoft). Data were analyzed as asingle factor design with single gene transformation as the main effect.Experimental units were the sum of peak areas of identifiedisoflavonoids normalized to dry weight. The mean square from the ANOVAwas used to calculate the least significant difference (LSD) for eachcomparison. The sum of isoflavonoid peak areas of samples from anon-transformed control line were compared with those of 25 independentpKS93s-transformed, hygromycin resistant lines. FIG. 25 shows a graphdepicting the distribution of the sum of isoflavone area per mg of dryweight of soybean somatic embryos transgenic for the isoflavone synthasegene and a control line. The results are depicted in the graph inascending order of the amount of total isoflavones produced. Some lines,such as the ones represented in bars 7 through 14, containedapproximately the same levels of isoflavones as the control line. Whilemost of the lines showed intermediate increases or decreases in theamounts of isoflavones produced, there are clear examples of lineshaving markedly increased or decreased amounts of isoflavones. Forexample, bar 25 represents a line which expresses 208% as muchisoflavones as the control line, bar 24 represents a line whichexpresses 184% as much isoflavones as the control line, and bar 1represents a line which produces only 25% of the isoflavones as thecontrol line. These differences in the amounts of isoflavones producedmay be caused by the position of the transgene in the chromosome, thenumber of copies of the gene that are integrated in the chromosome, DNAmethylation, gene silencing, etc. These results indicate that transgenicexpression of isoflavone synthase affords the ability to manipulateisoflavonoid levels as desired for a particular application; i.e.,transformants may be chosen for advancement that have large changes inisoflavonoid levels (i.e., very high as in IS19 or very low as in IS6)or more subtle changes in the content of isoflavonoids.

Example 16 Amplification and Analysis of Soybean Genomic IsoflavoneSynthase DNA

Genomic sequences encoding isoflavone synthase may be used to expressisoflavone synthase as well as the cDNA sequences. Therefore the genomicsequences containing the coding regions for the soybean isoflavonesynthase genes were isolated.

Soybean genomic DNA was prepared from Glycine max cv. Wye followingstandard protocols (DNeasy Plant Maxi Kit, Qiagen, Valencia, Calif.).Using this DNA as template, a genomic DNA fragment including thesequence corresponding to the soybean insert in sgs1c.pk006.o20 wasproduced by PCR with the primers listed as SEQ ID NO:41 and SEQ IDNO:42. A genomic DNA fragment including the sequence of CYP93C1 wasproduced with the primers listed as SEQ ID NO:7 and SEQ ID NO:51:5′-AAAATTAGCCTCACAAAAGCAAAG-3′ [SEQ ID NO:7]5′-GCAAACGAAGACAAATGGGAGATGATA3′ [SEQ ID NO:51]

Amplification was performed on a Perkin Elmer Applied Biosystems GeneAmpPCR System using the Expand™ Hi fidelity PCR system from BoehringerMannheim (Indianapolis, Ind.). These PCR fragments were cloned into thepCR2.1 vector (Invitrogen) and sequenced as described in Example 6. Thenucleotide sequence of the genomic fragment comprising the isoflavonesynthase sequence from clone sgs1c.pk006.o20 is given in SEQ ID NO:52.The nucleotide sequence of the genomic fragment comprising theisoflavone synthase sequence of CYP93C1 is given in SEQ ID NO:53. Bothgenes were found to contain one intron. The splice junction for bothintrons is within the codon for amino acid 300. The intron sequence inSEQ ID NO:52 corresponds to nucleotides 895 to 1112 (217 nucleotides),while the intron sequence in SEQ ID NO:53 corresponds to nucleotides 947to 1082 (135 nucleotides) in SEQ ID NO:53. Alignment of the intronnucleotide sequences using the Clustal method of alignment and thedefault parameters (KTUPLE 2, GAP PENALTY=5, WINDOW=4 and DIAGONALSSAVED=4) shows that the intron sequences are 46.3% identical.

Example 17 Alteration of Isoflavonoid Levels in Soybean Plants

The ability to alter the isoflavonoid levels in transgenic soybeanplants expressing the gene from soybean clone sgs1c.pk006.o20 was testedby transforming somatic embryo cultures with a vector containing thegene, allowing the plant to regenerate, and meassuring the levels ofisoflavonoids produced. In addition, the soybean IFS gene wastransformed in conjunction with the CRC gene.

Construction of Vectors for Transformation of Glycine max

A vector containing a chimeric isoflavone synthase gene was constructedas follows. The 1.6 Kb isoflavone synthase coding region from clonesgs1c.pk006.o20 (SEQ ID NO:1) was amplified using a standard PCRreaction in a GeneAmp PCR System using Pfu polymerase (Stratagene) withthe primers shown in SEQ ID NO:41 and SEQ ID NO:42 as in Example 9. Theplasmid pCW109 (World Patent Publication No. WO94/11516) was digestedwith Nco I. The resulting DNA fragments were treated with T4 DNApolymerase in the presence of dATP; dCTP, dGTP and dTTP to obtain bluntends followed by digestion with Kpn I. The ligation of these two DNAfragments created the plasmid pCW109—IFS, shown in FIG. 28, which hasoperably linked:

-   -   the beta-conglycinin promoter    -   the isoflavone synthase coding region    -   the phaseolin 3′ end

The 3.2 Kb fragment containing the beta-conglycinin/P-IFS-phaseolin 3′chimeric gene was purified from pCW109-IFS as a Hind III fragment andligated with Hind III-digested and phosphatase-treated pZBL102. pZBL102is derived from pKS18HH (described in U.S. Pat. No. 5,846,784) byreplacing the long Nos 3′ fragment in pKS18HH with the short Nos 3′fragment described in Example 13. The Sal I site between the twohygromycin phosphotransferase coding regions was deleted, and a Not Isite was added between the Hind III and Sal I sites 5′ to the 35Spromoter of the 35S-HPT gene.

The resulting plasmid, named pWSJ001, has a T7 promoter/HPT/T7terminator cassette for expression of the HPT enzyme in certain strainsof E. coli that are lysogenic for lambda DE3. The lambda DE3 carries theT7 RNA Polymerase gene under lacV5 control and is found in commerciallyavailable E. coli strains such as NovaBlue (DE3) (from Novagen). PlasmidpWSJ001 also contains the 35S/HPT/NOS 3′ cassette for constitutiveexpression of the HPT enzyme in plants. These two expression systemsallow selection for growth in the presence of hygromycin to be used as ameans of identifying cells that contain plasmid DNA sequences in bothbacterial and plant systems.

A vector containing a chimeric CRC gene was constructed as follows. Theplasmid pDP7951 of Example 13, FIG. 22, was digested with SmaI and thefragment containing the CRC coding region was purified. This CRCfragment was ligated to a modified vector containing the sequences ofpCW109 (World Patent Publication No. WO94/11516) with the substitutionof a phaseolin promoter fragment extending to −410 and including leadersequences to +77 (Slightom et al., 1991 Plant Mol Biol Man B16:1)instead of the beta-conglycinin promoter. Modification includeddigestion with NcoI and S1 nuclease treatment followed by religation toremove the ATG sequence of the NcoI site that follows the promoterfragment. The vector was then digested with KpnI and the ends filled inso that the SmaI CRC fragment was inserted in a blunt-end ligation. Fromthe resulting plasmid, the HindIII fragment containing the phaseolinpromoter-CRC-phaseolin 3′ chimeric gene was isolated and ligated withHindIII digested pZBL 102 (described above). The resulting plasmid wascalled pOY203.

Transformation of Somatic Soybean Embryo Cultures and Regeneration ofSoybean Plants

Soybean embryogenic suspension cultures were transformed with pWSJ001 orpWSJ001 in conjunction with pOY203 by the method of particle gunbombardment as in Example 15. Besides the media used for the soybeansomatic embryo cultures described in Example 15, the following mediawere used: Media SBP6 SB55 with only 0.5 mL 2,4-D SB71-1 (per liter) B5salts 1 ml B5 vitamin stock 30 g sucrose 750 mg MgCl2 2 g gelrite pH 5.7

Eleven days post bombardment, the liquid media was exchanged with freshSB55 containing 50 mg/mL hygromycin. The selective media was refreshedweekly. Seven weeks post bombardment, green, transformed tissue wasobserved growing from untransformed, necrotic embryogenic clusters.Isolated green tissue was removed and inoculated into individual flasksto generate new, clonally propagated, transformed embryogenic suspensioncultures. Thus each new line was treated as independent transformationevent. These suspensions can then be maintained as suspensions ofembryos clustered in an immature developmental stage through subcultureor regenerated into whole plants by maturation and germination ofindividual somatic embryos.

Transformed embryogenic clusters were removed from liquid culture andplaced on a solid agar media (SB103) containing no hormones orantibiotics. Embryos were cultured for eight weeks at 26° C. with mixedflorescent and incandescent lights on a 16:8 h day/night schedule.During this period, individual embryos were removed from the clustersand analyzed at various stages of embryo development. Selected lineswere assayed by PCR for the presence of the an additional IFS gene usingthe primers shown in SEQ ID NO:43 and SEQ ID NO:44. Separation of thePCR products on an agarose gel yielded a 1062 bp fragment indicative ofthe endogenous IFS gene (i.e., containing introns) and an 845 bpfragment in the embryos containing the transgene IFS. Somatic embryosbecome suitable for germination after eight weeks and were then removedfrom the maturation medium and dried in empty petri dishes for 1 to 5days. The dried embryos were then planted in SB71-1 medium where theywere allowed to germinate under the same lighting and germinationconditions described above. Germinated embryos were transferred tosterile soil and grown to maturity. Seed were harvested.

Seed from IFS-transformed and IFS+CRC-transformed soybean plants areanalyzed for isoflavonoid levels. Extracts are prepared and analyzed byHPLC as described in Example 15 except that a 150 to 200 mg chip ofsoybean seed is used for the analysis. Seeds with statisticallysignificant variation in the level of isoflavonoid concentration arefurther analyzed.

Various modifications of the invention in addition to those shown anddescribed herein will be apparent to those skilled in the art from theforegoing description. Such modifications are also intended to fallwithin the scope of the appended claims.

The disclosure of each reference set forth above is incorporated hereinby reference in its entirety.

1-19. (canceled)
 20. A plant comprising in its genome a chimericpolynucleotide comprising an isolated nucleic acid sequence encoding apolypeptide with isoflavone synthase activity having the amino acidsequence set forth in SEQ ID NO:66 wherein said chimeric polynucleotideis operably linked to at least one regulatory sequence.
 21. The plant ofclaim 20 further comprising in its genome a second chimeric sequencecomprising a nucleic acid sequence encoding a polypeptide that regulatesexpression of at least one enzyme of the phenylpropanoid pathway. 22.The plant of claim 20 wherein the plant is a soybean plant.
 23. Theplant of claim 20 wherein the plant is a corn plant.
 24. A seed from theplant of claim
 20. 25. A seed from the plant of claim
 21. 26-33.(canceled)
 34. A method of producing a plant with increased isoflavonoidcontent comprising (a) transforming a plant cell with a first chimericpolynucleotide comprising an isolated nucleic acid sequence encoding apolypeptide with isoflavone synthase activity having the amino acidsequence set forth in SEQ ID NO:66; (b) optionally transforming theplant cell with a second chimeric sequence comprising a nucleic acidsequence encoding a polypeptide that regulates expression of at leastone enzyme of the phenylpropanoid pathway; and (c) growing thetransformed plant cell under conditions that promote the regeneration ofa whole plant from the transformed cell wherein the transformed plantregenerated from the transformed cell produces an amount of anisoflavonoid that is greater than the amount of the isoflavonoid that isproduced in a plant that is regenerated from a plant cell that is nottransformed with the chimeric polynucleotide of part (a).
 35. The methodof claim 34 wherein the plant is a soybean plant.
 36. The method ofclaim 34 wherein the plant is a corn plant.
 37. The transgenic plantproduced by the method of claim
 34. 38. The transgenic plant of claim 37wherein the plant is a soybean plant.
 39. The transgenic plant of claim37 wherein the plant is a corn plant.
 40. A seed from the plant of claim37. 41-50. (canceled)